Growth Hormones with Prolonged In-Vivo Efficacy

ABSTRACT

The present invention relates to polypeptide compound optimized for subcutaneous administration, exemplified by growth hormone conjugates having a linker providing non-covalent binding to albumin.

FIELD OF THE INVENTION

The present invention relates to a growth hormone compound linked to analbumin binding residue via a hydrophilic spacer, and to methods ofpreparing and using such compounds. These compounds have a protractedprofile of action and are useful in therapy.

BACKGROUND OF THE INVENTION

It is well-known to modify the properties and characteristics ofpeptides by conjugating groups to the peptide which duly changes theproperties of the peptide. Such conjugation generally requires somefunctional group in the peptide to react with another functional groupin a conjugating group. Typically, amino groups, such as the N-terminalamino group or the ε-amino group in lysines, have been used incombination with a suitable acylating reagent such as described forGLP-1 in WO2005/027978. Alternatively, polyethylene glycol (PEG) orderivatives thereof may be attached to proteins. For a review, see Exp.Opion. Ther. Patent., 14, 859-894, (2004). It has been shown that theattachment of PEG to growth hormone may have a positive effect on theplasma half-life of growth hormone, WO 03/044056.

The use of carboxypeptidases to modify the C-terminal of peptides hasbeen described earlier. WO 92/05271 discloses the use ofcarboxypeptidases and nucleophilic compounds to amidate the C-terminalcarboxy group, and WO 98/38285 discloses variants of carboxypeptidase Yparticular suitable for this purpose.

EP 243 929 discloses the use of carboxypeptidase to incorporatepolypeptides, reporter groups or cytotoxic agents into the C-terminal ofproteins or polypeptides.

WO 2005/035553 describes methods for selective conjugation of peptidesby enzymatically incorporating a functional group at the C-terminal of apeptide.

Transglutaminase has previously been used to alter the properties ofpeptides. In the food industry and particular in the diary industry manytechniques are available to e.g. cross-bind peptides usingtransglutaminases. Other documents disclose the use of transglutaminaseto alter the properties of physiologically active peptides. EP 950665,EP 785276 and Sato, Adv. Drug Delivery Rev. 54, 487-504 (2002) disclosethe direct reaction between peptides comprising at least one Gln andamine-functionalised PEG or similar ligands in the presence oftransglutaminase, and Wada, Biotech. Lett. 23, 1367-1372 (2001)discloses the direct conjugation of β-lactoglobulin with fatty acids bymeans of transglutaminase. The international patent applicationpublished as WO 2005/070468 discloses the use of transglutaminase toincorporate a handle whereto conjugating groups can be attached.

Growth hormone is a key hormone involved in the regulation of not onlysomatic growth, but also in the regulation of metabolism of proteins,carbohydrates and lipids. The major effect of growth hormone is topromote growth. Human growth hormone is a 191 amino acid residue proteinwith the sequence:

(SEQ ID NO: 1)FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEG SCGF.

Administration of human growth hormone and closely related variantsthereof is used to treat a variety of growth hormone deficiency relateddiseases. Being a peptide, growth hormone is administered parenterally,i.e., by means of a needle. Growth hormone is, furthermore,characterised by a relative short half-life, hence frequentadministrations are required with the corresponding pain andinconvenience for the patient. Hence, there is still a need for theprovision of growth hormone compounds with improved pharmacologicalproperties, such as e.g. prolonged half-life.

The present invention provides novel growth hormone conjugates withimproved pharmacological properties as well as methods for theirproduction.

SUMMARY OF THE INVENTION

The bioavailability of a subcutaneously administered pharmaceuticalcompound may be related to the absorption rate. The ability of acompound to pass the tight junctions of the subcutaneous capillaries mayin part be related to their physical and chemical properties as well asthe molecular size or the hydrodynamic volume of the compound. A proteinconjugate such as a pegylated hGH (PEG-hGH) with a 40 kDa PEG has anapparent molecular weight of 150-250 kDa. A hGH molecule with covalentbound albumin has a molecular weight of 87 kDa, whereas a hGH moleculewith a non-covalent bound albumin will be dissociated from albumin partof the time and thus have a molecular weight of 22 kDa.

It is contemplated that the time spend in the dissociated state depends,at least partly, on the affinity of the albumin binding moiety. Thus theabsorption rate of a hGH molecule with a non-covalent bound albumin maybe faster than for a PEG-hGH. An increased rate of absorption may beobtained when using albumin binding moieties having lower affinity foralbumin.

Additionally, the physical and chemical properties of the linker and/orthe spacer providing the attachment of the albumin binding moiety to hGHwill influence the functionalities of the compounds.

The present inventors have surprisingly found that growth hormonecompounds (GH) may be selectively linked to an albumin bindingresidue—via a hydrophilic spacer that separates the GH and the albuminbinding residue with a chemical moiety having a mLogP<0—or a cLogP<0.5to obtain GH conjugates with improved pharmacological properties.

Furthermore, the present invention is based on the observation thatintroducing an albumin binding residue via a hydrophilic spacer in humangrowth hormone (hGH) can be done selectively wherein a large proportionof the biological activity can been retained. Preferably, an albuminbinding residue via a hydrophilic spacer is introduced at the positionscorresponding to positions phenylalanine 1, glutamine 40 and/orglutamine 141 in hGH having the sequence of SEQ ID No.1. The use oftransglutaminase (TGase), and in particular TGase fromStreptoverticillium mobaraenae or Streptomyces lydicus allows aselective introduction of an albumin binding residue via a hydrophilicspacer at positions 40 and/or 141, and the remaining 11 glutamineresidues are left untouched despite the fact that glutamine is asubstrate for transglutaminase.

Thus, in one embodiment of the present invention the growth hormonecompound (GH) is linked to one albumin binding residue via a hydrophilicspacer. Typically, the albumin binding residue is attached to theN-terminal, position 40 or position 141 of hGH via a hydrophilic spacer.In further embodiments two or three albumin binding residues areattached to the N-terminal, position 40 and/or position 141 of hGH via ahydrophilic spacer.

In a further embodiment of the present invention the hydrophilic spacerhas the formula

—X₁—X₂—X₃—X₄—

wherein

-   -   X₁ is        —W₁—[(CHR¹)_(I1)—W₂]_(m-1)—{[(CH₂)_(n1)E1]_(m2)-[(CHR²)_(I2)—W₃]_(m3)}_(n2)—,    -   X₂ is        —[(CHR³)_(I3)—W₄]_(m4)—{[(CH₂)_(n3)E2]_(m5)-[(CHR⁴)_(I4)—W₅]_(m6)}_(n4)—,    -   X₃ is —[(CHR⁵)₁₅—W₆]_(m7)—,    -   X₄ is —F-D1—(CH₂)_(I6)-D2-,    -   I1, I2, I3, I4, I5 and I6 independently are selected from 0-16,    -   m1, m3, m4, m6 and m7 independently are selected from 0-10,    -   m2 and m5 independently are selected from 0-25,    -   n1, n2, n3 and n4 independently are selected from 0-16,    -   F is aryl, hetaryl, pyrrolidine-2,5-dione or a valence bond,        wherein the aryl and hetaryl groups are optionally substituted        with halogen, —CN, —OH, —C(O)OH, —C(O)NH₂, —S(O)₂OH or        C₁₋₆-alkyl,    -   R¹, R², R³, R⁴ and R⁵ independently are selected from hydrogen,        —C(O)OH, —C(O)NH₂, —S(O)OH, —S(O)₂OH, —NH—C(═NH)—NH₂,        C₁₋₆-alkyl, aryl or hetaryl; wherein the alkyl, aryl and hetaryl        groups optionally are substituted with halogen, —C(O)OH,        —C(O)NH₂, —S(O)OH, —S(O)₂OH, —CN, or —OH,    -   D1, D2, E1 and E2 independently are selected from —O—, —NR⁶—,        —N(COR⁷)— or a valence bond; wherein R⁶ and R⁷ independently        represent hydrogen or C₁₋₆-alkyl,    -   W₁ to W₆ independently are selected from —C(O)NH—, —NHC(O)—,        —C(O)NHCH₂—, —CH₂NHC(O)—, —C(O)NHS(O)₂—, —S(O)₂NHC(O)—,        —OC(O)NH—, —NHC(O)O—, —C(O)CH₂—, —CH₂C(O)—, —C(O)CH═CH—,        —CH═CHC(O)—, —(CH₂)_(s1)—, —C(O)—, —C(O)O—, —OC(O)—, or a        valence bond; wherein s1 is 0 or 1.

It is a still further objective of the present invention to provide amethod for improving the properties of a GH by conjugation said proteinaccording to the methods of the present invention.

DEFINITIONS

In the present context, the term “growth hormone compound” as usedherein means growth hormone of mammalian origin, such as human, bovine,or porcine growth hormone, and recombinant growth hormone, such asrecombinant human, bovine, or porcine growth hormone, and variants ofsuch growth hormones. As used herein “GH” and “growth hormone compound”are interchangeable. When GH is a variant of growth hormone of mammalianorigin, such as hGH and recombinant hGH, said variant is understood tobe the compound obtained by substituting one or more amino acid residuesin the growth hormone, e.g. hGH, sequence with another natural orunnatural amino acid; and/or by adding one or more natural or unnaturalamino acids to the growth hormone, e.g. hGH, sequence; and/or bydeleting one or more amino acid residue from the growth hormone, e.g.hGH, sequence, wherein any of these steps may optionally be followed byfurther derivatization of one or more amino acid residues. Inparticular, such substitutions are conservative in the sense that oneamino acid residue is substituted by another amino acid residue from thesame group, i.e. by another amino acid residue with similar properties.Amino acids may conveniently be divided in the following groups based ontheir properties: Basic amino acids (such as arginine, lysine,histidine), acidic amino acids (such as glutamic acid and asparticacid), polar amino acids (such as glutamine, cysteine and asparagine),hydrophobic amino acids (such as leucine, isoleucine, proline,methionine and valine), aromatic amino acids (such as phenylalanine,tryptophan, tyrosine) and small amino acids (such as glycine, alanine,serine and threonine). Typically, the GH has at least 80% identity withhGH, and typically, has at least 20% of the growth hormone activity ofhGH as determined in assay I herein.

In the present context, the term “albumin binding residue” as usedherein means a residue which binds noncovalently to human serum albumin.The albumin binding residue attached to the growth hormone compound (GH)typically has a binding affinity towards human serum albumin that isbelow about 10 μM or even below about 1 μM. A range of albumin bindingresidues are known among linear and branched lipohophillic moietiescontaining 12-40 carbon atoms, compounds with a cyclopentanophenanthreneskeleton, and/or peptides having 10-45 amino acid residues etc. Albuminbinding properties can be measured by surface plasmon resonance asdescribed in J. Biol. Chem. 277(38), 35035-35042, (2002).

The term “hydrophilic spacer” as used herein means a spacer thatseparates a growth hormone compound and an albumin binding residue witha chemical moiety which comprises at least 5 nonhydrogen atoms where30-50% of these are either N or O.

In the present context, the term “transamination” and related terms areintended to indicate a reaction wherein the amide nitrogen in the sidechain of glutamine is exchanged with nitrogen from another compound, inparticular nitrogen from another nitrogen containing nucleophile.

Transglutaminase (E.C.2.3.2.13) is also known asprotein-glutamine-γ-glutamyltransferase and catalyses the generalreaction

Q—C(O)—NH₂ (amine acceptor) may represent a glutamine residue containingpeptide or protein and Q′—NH₂ (amine donor) represents anamine-containing nucleophile. Alternatively, Q—C(O)—NH₂ and Q′—NH₂ mayrepresent an amine acceptor and a lysine-containing peptide or protein,respectively. In the present invention, however, Q—C(O)—NH₂ represents aglutamine residue containing growth hormone and Q′—NH₂ represents anamine-containing nucleophile as indicated above.

Examples of useful transglutaminases include microbialtransglutaminases, such as e.g. those from Streptomyces mobaraense,Streptomyces cinnamoneum and Streptomyces griseocarneum (all disclosedin U.S. Pat. No. 5,156,956, which is incorporated herein by reference),and from Streptomyces lavendulae (disclosed in U.S. Pat. No. 5,252,469,which is incorporated herein by reference) and Streptomyces ladakanum(JP 2003/199569, which is incorporated herein by reference). It shouldbe noted that members of the former genus Streptoverticillium are nowincluded in the genus Streptomyces (Kaempfer, J. Gen. Microbiol. 137,1831-1892 (1991)). Other useful microbial transglutaminases have beenisolated from Bacillus subtilis (disclosed in U.S. Pat. No. 5,731,183,which is incorporated herein by reference) and from various Myxomycetes.Other examples of useful microbial transglutaminases are those disclosedin WO 96/06931 (e.g. transglutaminase from Bacillus lydicus) and WO96/22366, both of which are incorporated herein by reference. Usefulnon-microbial transglutaminases include guinea-pig livertransglutaminase, and transglutaminases from various marine sources likethe flat fish Pagrus major (disclosed in EP-0555649, which isincorporated herein by reference), and the Japanese oyster Crassostreagigas (disclosed in U.S. Pat. No. 5,736,356, which is incorporatedherein by reference).

In the present context, the term “not accessible” is intended toindicate that something is absent or de facto absent in the sense thatit cannot be reached. When it is stated that functional groups are notaccessible in a protein to be conjugated it is intended to indicate thatsaid functional group is absent from the protein or, if present, in someway prevented from taking part in reactions. By way of example, saidfunctional group could be buried deep in the structure of the protein sothat it is shielded from participating in the reaction. It is recognisedthat whether or not a functional group is accessible depends on thereaction conditions. It may be envisaged that, e.g. in the presence ofdenaturing agents or at elevated temperatures the protein may unfold toexpose otherwise not accessible functional groups. It is to beunderstood that “not accessible” means “not accessible at the reactioncondition chosen for the particular reaction of interest”.

The term “alkane” or “alkyl” is intended to indicate a saturated,linear, branched and/or cyclic hydrocarbon. Unless specified withanother number of carbon atoms, the term is intended to indicatehydrocarbons with from 1 to 30 (both included) carbon atoms, such as 1to 20 (both included), such as from 1 to 10 (both included), e.g. from 1to 5 (both included). The terms alkyl and alkylene refer to thecorresponding radical and bi-radical, respectively.

The term “C₁₋₆ alkyl” refers to a straight chained or branched saturatedhydrocarbon having from one to six carbon atoms inclusive. Examples ofsuch groups include, but are not limited to, methyl, 2-propyl, 1-butyl,2-butyl, 2-methyl-2-propyl, 2-methyl-1-butyl and n-hexyl.

The term “C₃₋₁₀ cycloalkyl” typically refers to cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, andcyclodecanyl.

The term “alkene” is intended to indicate linear, branched and/or cyclichydrocarbons comprising at least one carbon-carbon double bond. Unlessspecified with another number of carbon atoms, the term is intended toindicate hydrocarbons with from 2 to 30 (both included) carbon atoms,such as 2 to 20 (both included), such as from 2 to 10 (both included),e.g. from 2 to 5 (both included). The terms alkenyl and alkenylene referto the corresponding radical and bi-radical, respectively.

The term “alkyne” is intended to indicate linear, branched and/or cyclichydrocarbons comprising at least one carbon-carbon triple bond, and itmay optionally comprise one or more carbon-carbon double bonds. Unlessspecified with another number of carbon atoms, the term is intended toindicate hydrocarbons with from 2 to 30 (both included) carbon atoms,such as from 2 to 20 (both included), such as from 2 to 10 (bothincluded), e.g. from 2 to 5 (both included). The terms alkynyl andalkynylene refer to the corresponding radical and bi-radical,respectively.

The term “homocyclic aromatic compound” is intended to indicate aromatichydrocarbons, such as benzene and naphthalene.

The term “heterocyclic compound” is intended to indicate a cycliccompound comprising 5, 6 or 7 ring atoms from which 1, 2, 3 or 4 arehetero atoms selected from N, O and/or S. Examples include heterocyclicaromatic compounds, such as thiophene, furan, pyran, pyrrole, imidazole,pyrazole, isothiazole, isooxazole, pyridine, pyrazine, pyrimidine,pyridazine, as well as their partly or fully hydrogenated equivalents,such as piperidine, pirazolidine, pyrrolidine, pyroline, imidazolidine,imidazoline, piperazine and morpholine.

The terms “hetero alkane”, “hetero alkene” and “hetero alkyne” areintended to indicate alkanes, alkenes and alkynes as defined above, inwhich one or more hetero atom or group have been inserted into thestructure of said moieties. Examples of hetero groups and atoms include—O—, —S—, —S(O)—, —S(O)₂—, —C(O)—C(S)— and —N(R*)—, wherein R*represents hydrogen or C₁-C₆-alkyl. Examples of heteroalkanes include.

The term “radical” or “biradical” is intended to indicate a compoundfrom which one or two, respectively, hydrogen atoms have been removed.When specifically stated, a radical may also indicate the moiety formedby the formal removal of a larger group of atoms, e.g. hydroxyl, from acompound.

The term “halogen” is intended to indicate members of the seventh maingroup of the periodic table, e.g. F, Cl, Br and I.

In the present context, the term “aryl” is intended to indicate acarbocyclic aromatic ring radical or a fused aromatic ring systemradical wherein at least one of the rings are aromatic. Typical arylgroups include phenyl, biphenylyl, naphthyl, and the like.

The term “heteroaryl” or “hetaryl”, as used herein, alone or incombination, refers to an aromatic ring radical with for instance 5 to 7member atoms, or to a fused aromatic ring system radical with forinstance from 7 to 18 member atoms, wherein at least one ring isaromatic, containing one or more heteroatoms as ring atoms selected fromnitrogen, oxygen, or sulfur heteroatoms, wherein N-oxides and sulfurmonoxides and sulfur dioxides are permissible heteroaromaticsubstitutions. Examples include furanyl, thienyl, thiophenyl, pyrrolyl,imidazolyl, pyrazolyl, triazolyl, tetrazolyl, thiazolyl, oxazolyl,isoxazolyl, oxadiazolyl, thiadiazolyl, isothiazolyl, pyridinyl,pyridazinyl, pyrazinyl, pyrimidinyl, quinolinyl, isoquinolinyl,benzofuranyl, benzothiophenyl, indolyl, and indazolyl, and the like.

The term “conjugate” as a noun is intended to indicate a modifiedprotein, i.e. a protein with a moiety bonded to it in order to modifythe properties of said protein. As a verb, the term is intended toindicate the process of bonding a moiety to a protein to modify theproperties of said protein.

As used herein, the term “prodrug” indicates biohydrolyzable amides andbiohydrolyzable esters and also encompasses a) compounds in which thebiohydrolyzable functionality in such a prodrug is encompassed in thecompound according to the present invention, and b) compounds which maybe oxidized or reduced biologically at a given functional group to yielddrug substances according to the present invention. Examples of thesefunctional groups include 1,4-dihydropyridine,N-alkylcarbonyl-1,4-dihydropyridine, 1,4-cyclohexadiene, tert-butyl, andthe like.

As used herein, the term “biohydrolyzable ester” is an ester of a drugsubstance (in casu, a compound according to the invention) which eithera) does not interfere with the biological activity of the parentsubstance but confers on that substance advantageous properties in vivosuch as duration of action, onset of action, and the like, or b) isbiologically inactive but is readily converted in vivo by the subject tothe biologically active principle. The advantage is, for exampleincreased solubility or that the biohydrolyzable ester is orallyabsorbed from the gut and is transformed to a compound according to thepresent invention in plasma. Many examples of such are known in the artand include by way of example lower alkyl esters (e.g., C₁-C₄), loweracyloxyalkyl esters, lower alkoxyacyloxyalkyl esters, alkoxyacyloxyesters, alkyl acylamino alkyl esters, and choline esters.

As used herein, the term “biohydrolyzable amide” is an amide of a drugsubstance (in casu, a compound according to the present invention) whicheither a) does not interfere with the biological activity of the parentsubstance but confers on that substance advantageous properties in vivosuch as duration of action, onset of action, and the like, or b) isbiologically inactive but is readily converted in vivo by the subject tothe biologically active principle. The advantage is, for exampleincreased solubility or that the biohydrolyzable amide is orallyabsorbed from the gut and is transformed to a compound according to thepresent invention in plasma. Many examples of such are known in the artand include by way of example lower alkyl amides, a-amino acid amides,alkoxyacyl amides, and alkylaminoalkylcarbony) amides.

In the present context, the term “pharmaceutically acceptable salt” isintended to indicate salts which are not harmful to the patient. Suchsalts include pharmaceutically acceptable acid addition salts,pharmaceutically acceptable metal salts, ammonium and alkylated ammoniumsalts. Acid addition salts include salts of inorganic acids as well asorganic acids. Representative examples of suitable inorganic acidsinclude hydrochloric, hydrobromic, hydroiodic, phosphoric, sulfuric,nitric acids and the like. Representative examples of suitable organicacids include formic, acetic, trichloroacetic, trifluoroacetic,propionic, benzoic, cinnamic, citric, fumaric, glycolic, lactic, maleic,malic, malonic, mandelic, oxalic, picric, pyruvic, salicylic, succinic,methanesulfonic, ethanesulfonic, tartaric, ascorbic, pamoic,bismethylene salicylic, ethanedisulfonic, gluconic, citraconic,aspartic, stearic, palmitic, EDTA, glycolic, p-aminobenzoic, glutamic,benzenesulfonic, p-toluenesulfonic acids and the like. Further examplesof pharmaceutically acceptable inorganic or organic acid addition saltsinclude the pharmaceutically acceptable salts listed in J. Pharm. Sci.66, 2, (1977) which is incorporated herein by reference. Examples ofmetal salts include lithium, sodium, potassium, magnesium salts and thelike. Examples of ammonium and alkylated ammonium salts includeammonium, methylammonium, dimethylammonium, trimethylammonium,ethylammonium, hydroxyethylammonium, diethylammonium, butylammonium,tetramethylammonium salts and the like.

A “therapeutically effective amount” of a compound as used herein meansan amount sufficient to cure, alleviate or partially arrest the clinicalmanifestations of a given disease and its complications. An amountadequate to accomplish this is defined as “therapeutically effectiveamount”. Effective amounts for each purpose will depend on the severityof the disease or injury as well as the weight and general state of thesubject. It will be understood that determining an appropriate dosagemay be achieved using routine experimentation, by constructing a matrixof values and testing different points in the matrix, which is allwithin the ordinary skills of a trained physician or veterinary.

The term “treatment” and “treating” as used herein means the managementand care of a patient for the purpose of combating a condition, such asa disease or a disorder. The term is intended to include the fullspectrum of treatments for a given condition from which the patient issuffering, such as administration of the active compound to alleviatethe symptoms or complications, to delay the progression of the disease,disorder or condition, to alleviate or relief the symptoms andcomplications, and/or to cure or eliminate the disease, disorder orcondition as well as to prevent the condition, wherein prevention is tobe understood as the management and care of a patient for the purpose ofcombating the disease, condition, or disorder and includes theadministration of the active compounds to prevent the onset of thesymptoms or complications. The patient to be treated is preferably amammal; in particular a human being, but it may also include animals,such as dogs, cats, cows, sheep and pigs.

DESCRIPTION OF THE INVENTION

In its broadest aspect the present invention relates to a growth hormoneconjugate which comprises a growth hormone compound (GH) linkedselectively to an albumin binding residue via a hydrophilic spacer, or apharmaceutically acceptable salt, solvate or prodrug thereof.

In one embodiment of the present invention the hydrophilic spacer has amLogP<0.

Solubility of a hydrophilic spacer can be described by its logP value.LogP, also known as the partition coefficient, is the logarithm of theratio of concentrations of a compound in the two phases of a mixture oftwo immiscible solvents at equilibrium. Typically one of the solvents iswater while the second is selected from octan-1-ol, chloroform,cyclohexane and propylene glycol dipelargonate (PGDP). LogP valuesmeasured in these different solvents show differences principally due tohydrogen bonding effects. Octanol can donate and accept hydrogen bondswhereas cyclohexane is inert. Chloroform can donate hydrogen bondswhereas PGDP can only accept them.

In another embodiment of the invention, the hydrophilic spacer has aLogP of below −0.5 in either octan-1-ol, chloroform, cyclohexane andpropylene glycol dipelargonate (PGDP).

In a further embodiment, the hydrophilic spacer has a logP below -1 ineither octan1-ol, chloroform, cyclohexane and propylene glycoldipelargonate (PGDP).

Alternatively, the LogP value can be calculated as mLogP and/or cLogPfor the albumin binder part or hydrophilic spacer part using publishedalgorithms (J. Am. Chem. Soc., 86 (1964) 5175-5180 “A New SubstituentConstant, ∉, Derived from Partition Coefficients”, C. A. Lipinski et al.Advanced Drug Delivery Reviews, 23 (1997) 3-25, “Experimental andComputational Approaches to Estimate Solubility and Permeability in DrugDiscovery and Development Settings” and I. Moriguchi, S. Hirono, I.Nakagome, H. Hirano, Chem. and Pharm. Bull., 42 (1994) 976-978“Comparison of Reliability of logP Values for Drugs Calculated bySeveral Methods”. In one embodiment of the present invention thehydrophilic spacer has a cLogP<0.5.

In a further embodiment the growth hormone compound (GH) is linked toone albumin binding residue via a hydrophilic spacer.

In another embodiment the growth hormone compound (GH) is linked to twoalbumin binding residues via one or two hydrophilic spacer(s). Thus, inone example one albumin binding residue is linked via one hydrophilicspacer to glutamine in position 40 and the other albumin binding residueis linked via one hydrophilic spacer to glutamine in position 141; oralternatively two albumin binding residues are linked via onehydrophilic spacer to glutamine in position 40 or 141 or the N-terminal.In a still other embodiment the growth hormone compound (GH) is linkedto three albumin binding residues via one or more hydrophilic spacer(s).

In an embodiment the hydrophilic spacer comprise at least one OEG motif,the radical 8-amino-3,6-dioxaoctanic acid, i.e.—NH—(CH₂)₂—O—(CH₂)₂—O—CH₂—C(O)—. In further specified embodiment thehydrophilic spacer comprise at least two OEG motifs. The orientation ofsuch OEG motif(s) is in one embodiment so that the —C(O)— is closest tothe growth hormone compound but not connecting the growth hormonecompound and the albumin binder linker and the —NH— is closest to thealbumin binding residue. In additional embodiments comprising two OEGmotifs the two motifs have identical orientation or differentorientation. In an embodiment two such OEG motifs are located adjacentto each other whereas in alternative embodiments such OEG motifs areserrated by one or more atoms covalently linked.

In an embodiment the hydrophilic spacer comprise at lease one glutamicacid residue. The amino acid glutamic acid comprises two carboxylic acidgroups. Its gamma-carboxy group may be used for forming an amide bondwith the epsilon-amino group of lysine, or with an amino group of an OEGmolecule, if present, or with the amino group of another Glu residue, ifpresent. The alfa-carboxy group may alternatively be used for formingsimilar amide bond with the epsilon-amino group of lysine, or with anamino group of an OEG molecule, if present, or with the amino group ofanother Glu residue, if present. The amino group of Glu may in turn forman amide bond with the carboxy group of the albumin binding residue, orwith the carboxy group of an OEG motif, if present, or with thegamma-carboxy group or alfa carboxy group of another Glu, if present.The linkage of the amino group of one Glu to a gamma carboxy group of asecond Glu may be referred to as a “gamma-Glu” motif.

In an embodiment the hydrophilic spacer comprise at lease one combinedOEG-Glu motif (—NH—(CH₂)₂—O—(CH₂)₂—O—CH₂—C(O)NH-CH(C(O)OH)—(CH₂)₂—C(O)—)or at least one combined Glu-OEG motif(—NH—CH(C(O)OH)—(CH₂)₂—C(O)NH—(CH₂)₂—O—(CH₂)₂—O—CH₂—C(O)—) orcombination here of, where in such Glu-OEG and OEG-Glu motifs may beseparated by one or more covalently linked atoms or directly bond toeach other by an amide bond of the Glu's forming a gamma-Glu.

In a still further embodiment of the growth hormone conjugate thehydrophilic spacer has the formula

—X₁—X₂—X₃—X₄—

wherein

-   -   X₁ is        —W₁—[(CHR¹)_(I1)—W₂]_(m-1)—{[(CH₂)_(n1)E1]_(m2)-[(CHR²)_(I2)—W₃]_(m3)}_(n2)—,    -   X₂ is        —[(CHR³)_(I3)—W₄]_(m4)—{[(CH₂)_(n3)E2]_(m5)-[(CHR⁴)_(I4)—W₅]_(m6)}_(n4)—,    -   X₃ is —[(CHR⁵)₁₅—W₆]_(m7)—,    -   X₄ is —F-D1—(CH₂)_(I6)-D2-,    -   I1, I2, I3, I4, I5 and I6 independently are selected from 0-16,    -   m1, m3, m4, m6 and m7 independently are selected from 0-10,    -   m2 and m5 independently are selected from 0-25,    -   n1, n2, n3 and n4 independently are selected from 0-16,    -   F is aryl, hetaryl, pyrrolidine-2,5-dione or a valence bond,        wherein the aryl and hetaryl groups are optionally substituted        with halogen, —CN, —OH, —C(O)OH, —C(O)NH₂, —S(O)₂OH or        C₁₋₆-alkyl,    -   R¹, R², R³, R⁴ and R⁵ independently are selected from hydrogen,        —C(O)OH, —C(O)NH₂, —S(O)OH, —S(O)₂OH, —NH—C(═NH)—NH₂,        C₁₋₆-alkyl, aryl or hetaryl; wherein the alkyl, aryl and hetaryl        groups optionally are substituted with halogen, —C(O)OH,        —C(O)NH₂, —S(O)OH, —S(O)₂OH, —CN, or —OH,    -   D1, D2, E1 and E2 independently are selected from —O—, —NR⁶—,        —N(COR⁷)— or a valence bond; wherein R⁶ and R⁷ independently        represent hydrogen or C₁₋₆-alkyl,    -   W₁ to W₆ independently are selected from —C(O)NH—, —NHC(O)—,        —C(O)NHCH₂—, —CH₂NHC(O)—, —C(O)NHS(O)₂—, —S(O)₂NHC(O)—,        —OC(O)NH—, —NHC(O)O—, —C(O)CH₂—, —CH₂C(O)—, —C(O)CH═CH—,        —CH═CHC(O)—, —(CH₂)_(s1)—, —C(O)—, —C(O)O—, —OC(O)—, or a        valence bond; wherein s1 is 0 or 1.

In an embodiment D1 is selected from —O—, —NR⁶—, —N(COR⁷)— or a valencebond, D2 is —NR⁶—, E1 and E2 are independently selected from —O—, —NR⁶—,—N(COR⁷)— or a valence bond and R⁶ and R⁷ independently representhydrogen or C₁₋₆-alkyl,

In a further aspect the present invention relates to a growth hormoneconjugate of the formula (I):

A-W—B-GH   (I)

wherein

GH represents a growth hormone compound,

B represents a hydrophilic spacer,

W is a chemical group linking A and B, and

A represent an albumin binding residue; and

pharmaceutically acceptable salts, solvates and prodrugs thereof.

As described above the growth hormone compound (GH) may be linked to twoalbumin binding residues via a hydrophilic spacer, accordingly in astill further aspect the present invention relates to a growth hormoneconjugate of the formula (II):

A-W—B-GH—B′—W′-A′  (II)

wherein

GH represents a growth hormone compound,

W is a chemical group linking A and B,

W′ is a chemical group linking A′ and B′,

A and A′ independently represents an albumin binding residue,

B and B′ independently are hydrophilic spacers, and

pharmaceutically acceptable salts, solvates and prodrugs thereof.

In the conjugate of formula (II) W′ is selected from the same groups asW, A′ is selected from the same groups as A and B′ is selected from thesame groups as B, and it should be understood that W and W′, A and A′,and B and B′ are independently selected from any one of the respectivegroups as defined hereunder. Thus, any embodiments of W, A, and Bhereunder are also embodiments of W′, A′, and B′. Furthermore, any oneof the embodiments described herein refers independently to both of theconjugates of formula (I) and (II), as well as the broad aspect andembodiments thereof when suitable.

The above embodiments as well as the embodiments to be describedhereunder should be seen as referring to any one of the aspectsdescribed herein as well as any one of the embodiments described hereinunless it is specified that an embodiment relates to a certain aspect oraspects of the present invention.

In one embodiment GH is a variant of hGH, wherein a variant isunderstood to be the compound obtained by substituting one or more aminoacid residues in the hGH sequence with another natural or unnaturalamino acid; and/or by adding one or more natural or unnatural aminoacids to the hGH sequence; and/or by deleting one or more amino acidresidue from the hGH sequence, wherein any of these steps may optionallybe followed by further derivatization of one or more amino acidresidues. In particular, such substitutions are conservative in thesense that one amino acid residue is substituted by another amino acidresidue from the same group, i.e. by another amino acid residue withsimilar properties. Amino acids may conveniently be divided in thefollowing groups based on their properties: Basic amino acids (such asarginine, lysine, histidine), acidic amino acids (such as glutamic acidand aspartic acid), polar amino acids (such as glutamine, cysteine andasparagine), hydrophobic amino acids (such as leucine, isoleucine,proline, methionine and valine), aromatic amino acids (such asphenylalanine, tryptophan, tyrosine) and small amino acids (such asglycine, alanine, serine and threonine.).

In a further embodiment GH represents a growth hormone compoundcomprising an amino acid sequence having at least 90% identity to theamino acid sequence of human growth hormone (hGH) (SEQ ID NO:1). Infurther embodiments, GH has at least 80%, such as at least 85%, such asat least 95% identity with hGH. In further embodiments, said identitiesto hGH is coupled to at least 20%, such as at least 40%, such as atleast 60%, such as at least 80% of the growth hormone activity of hGH asdetermined in assay I herein. Any one of the sequence identityembodiments may be combined with any one of the activity embodiments ,such as a GH having at least 80% identity with hGH and coupled to atleast 60% of the growth hormone activity of hGH; a GH having at least90% identity with hGH and coupled to at least 40% of the growth hormoneactivity of hGH; a GH having at least 95% identity with hGH and coupledto at least 80% of the growth hormone activity of hGH, and so forth.

In a still further embodiment GH is hGH (SEQ ID NO:1).

In a further embodiment of the conjugate of formula (I) or (II), thehydrophilic spacer B has the formula

—X₁—X₂—X₃—X₄—

wherein

-   -   X₁ is        —W₁—[(CHR¹)_(I1)—W₂]_(m-1)—{[(CH₂)_(n1)E1]_(m2)-[(CHR²)_(I2)—W₃]_(m3)}_(n2)—,    -   X₂ is        —[(CHR³)_(I3)—W₄]_(m4)—{[(CH₂)_(n3)E2]_(m5)-[(CHR⁴)_(I4)—W₅]_(m6)}_(n4)—,    -   X₃ is —[(CHR⁵)_(I5)—W₆]_(m7)—,    -   X₄ is —F-D1—(CH₂)_(I6)-D2-,    -   I1, I2, I3, I4, I5 and I6 independently are selected from 0-16,    -   m1, m3, m4, m6 and m7 independently are selected from 0-10,    -   m2 and m5 independently are selected from 0-25,    -   n1, n2, n3 and n4 independently are selected from 0-16,    -   F is aryl, hetaryl, pyrrolidine-2,5-dione or a valence bond,        wherein the aryl and hetaryl groups are optionally substituted        with halogen, —CN, —OH, —C(O)OH, —C(O)NH₂, —S(O)₂OH or        C₁₋₆-alkyl,    -   R¹, R², R³, R⁴ and R⁵ independently are selected from hydrogen,        —C(O)OH, —C(O)NH₂, —S(O)OH, —S(O)₂OH, —NH—C(═NH)—NH₂,        C₁₋₆-alkyl, aryl or hetaryl; wherein the alkyl, aryl and hetaryl        groups optionally are substituted with halogen, —C(O)OH,        —C(O)NH₂, —S(O)OH, —S(O)₂OH, —CN, or —OH,    -   D1, D2, E1 and E2 independently are selected from —O—, —NR⁶—,        —N(COR⁷)— or a valence bond; wherein R⁶ and R⁷ independently        represent hydrogen or C₁₋₆-alkyl,    -   W₁ to W₆ independently are selected from —C(O)NH—, —NHC(O)—,        —C(O)NHCH₂—, —CH₂NHC(O)—, —C(O)NHS(O)₂—, —S(O)₂NHC(O)—,        —OC(O)NH—, —NHC(O)O—, —C(O)CH₂—, —CH₂C(O)—, —C(O)CH═CH—,        —CH═CHC(O)—, —(CH₂)_(s2)—, —C(O)—, —C(O)O—, —OC(O)—, or a        valence bond; wherein s2 is 0 or 1.

In a further embodiment W₁ is selected from —C(O)NH—, —NHC(O)—,—C(O)NHCH₂—, —CH₂NHC(O)—, —C(O)NHS(O)₂—, —S(O)₂NHC(O)— or a valencebond. Typically, W₁ is selected from —C(O)NH—, —NHC(O)—, —C(O)NHS(O)₂—,

In a further embodiment W₂ is selected from —C(O)NH—, —NHC(O)—,—C(O)NHCH₂—, —CH₂NHC(O)—, —C(O)NHS(O)₂—, —S(O)₂NHC(O)— or a valencebond. Typically, W₂ is selected from —C(O)NH—, —NHC(O)—, —C(O)NHS(O)₂—,

In a further embodiment W₃ is selected from —C(O)NH—, —NHC(O)—,—C(O)NHCH₂—, —CH₂NHC(O)—, —C(O)NHS(O)₂—, —S(O)₂NHC(O)— or a valencebond. Typically, W₃ is selected from —C(O)NH—, —NHC(O)—, —C(O)NHS(O)₂—,

In a further embodiment W₄ is selected from —C(O)NH—, —NHC(O)—,—C(O)NHCH₂—, —CH₂NHC(O)—, —C(O)NHS(O)₂—, —S(O)₂NHC(O)— or a valencebond. Typically, W₄ is selected from —C(O)NH—, —NHC(O)—, —C(O)NHS(O)₂—,

In a further embodiment W₅ is selected from —C(O)NH—, —NHC(O)—,—C(O)NHCH₂—, —CH₂NHC(O)—, —C(O)NHS(O)₂—, —S(O)₂NHC(O)— or a valencebond. Typically, W₅ is selected from —C(O)NH—, —NHC(O)—, —C(O)NHS(O)₂—,

In a further embodiment W₆ is selected from —C(O)NH—, —NHC(O)—,—C(O)NHCH₂—, —CH₂NHC(O)—, —C(O)NHS(O)₂—, —S(O)₂NHC(O)— or a valencebond. Typically, W₆ is selected from —C(O)NH—, —NHC(O)—, —C(O)NHS(O)₂—,

In a further embodiment R¹ selected from hydrogen, —C(O)OH, —C(O)NH₂,—S(O)₂OH or C₁₋₆-alkyl; wherein the alkyl group optionally issubstituted with —C(O)OH, —C(O)NH₂, —S(O)₂OH. Typically, R¹ is selectedfrom —C(O)OH, —C(O)NH₂, or C₁₋₆-alkyl; wherein the alkyl groupoptionally is substituted with —C(O)OH, —C(O)NH₂, or —S(O)₂OH.

In a further embodiment R² is selected from hydrogen, —C(O)OH, —C(O)NH₂,—S(O)₂OH or C₁₋₆-alkyl; wherein the alkyl group optionally issubstituted with —C(O)OH, —C(O)NH₂, —S(O)₂OH. Typically, R² is selectedfrom —C(O)OH, —C(O)NH₂, or C₁₋₆-alkyl; wherein the alkyl groupoptionally is substituted with —C(O)OH, —C(O)NH₂, or —S(O)₂OH.

In a further embodiment R³ is selected from hydrogen, —C(O)OH, —C(O)NH₂,—S(O)₂OH or C₁₋₆-alkyl; wherein the alkyl group optionally issubstituted with —C(O)OH, —C(O)NH₂, —S(O)₂OH. Typically, R³ is selectedfrom —C(O)OH, —C(O)NH₂, or C₁₋₆-alkyl; wherein the alkyl groupoptionally is substituted with —C(O)OH, —C(O)NH₂, or —S(O)₂OH.

In a further embodiment R⁴ is selected from hydrogen, —C(O)OH, —C(O)NH₂,—S(O)₂OH or C₁₋₆-alkyl; wherein the alkyl group optionally issubstituted with —C(O)OH, —C(O)NH₂, —S(O)₂OH. Typically, R⁴ is selectedfrom —C(O)OH, —C(O)NH₂, or C₁₋₆-alkyl; wherein the alkyl groupoptionally is substituted with —C(O)OH, —C(O)NH₂, or —S(O)₂OH.

In a further embodiment R⁵ is selected from hydrogen, —C(O)OH, —C(O)NH₂,—S(O)₂OH or C₁₋₆-alkyl; wherein the alkyl group optionally issubstituted with —C(O)OH, —C(O)NH₂, —S(O)₂OH. Typically, R⁵ is selectedfrom —C(O)OH, —C(O)NH₂, or C₁₋₆-alkyl; wherein the alkyl groupoptionally is substituted with —C(O)OH, —C(O)NH₂, or —S(O)₂OH.

In a further embodiment E1 and E2 independently are selected from —O—,—NR⁶—, —N—(COR⁷)— or a valence bond. R⁶ and R⁷ independently representhydrogen or C₁₋₆-alkyl.

In a further embodiment E1 is selected from —O— or —NR⁶— or a valencebond. Typically, E1 is selected from —O—.

In a further embodiment E2 is selected from —O— or —NR⁶— or a valencebond. Typically, E2 is selected from —O—.

In a further embodiment E1 and E2 are both —O—.

In a further embodiment E1 and E2 are both —NR⁶—.

In a further embodiment F is phenyl, pyrrolidine-2,5-dione or a valencebond.

In a further embodiment D1 is selected from —O—, —NR⁶—, —N(COR⁷)— or avalence bond.

In a further embodiment D1 is selected from —O— or —NR⁶— or a valencebond. Typically, D1 is selected from —NR⁶—.

In a further embodiment D2 is selected from —O—, —NR⁶—, —N(COR⁷)— or avalence bond.

In a further embodiment D2 is selected from —O— or —NR⁶— or a valencebond. Typically, D2 is selected from —NR⁶—.

R⁶ and R⁷ independently represent hydrogen or C₁₋₆-alkyl,

In a further embodiment I1 is 0-6, such as 0, 1, 2, 3, 4, 5 or 6.

In a further embodiment I2 is 0-6, such as 0, 1, 2, 3, 4, 5 or 6.

In a further embodiment I3 is 0-6, such as 0, 1, 2, 3, 4, 5 or 6.

In a further embodiment I4 is 0-6, such as 0, 1, 2, 3, 4, 5 or 6.

In a further embodiment I5 is 0-6, such as 0, 1, 2, 3, 4, 5 or 6.

In a further embodiment I6 is 0-6, such as 0, 1, 2, 3, 4, 5 or 6.

In a further embodiment m1 is 0-6, such as 0, 1, 2, 3, 4, 5 or 6.

In a further embodiment m2 is 0-10, such as 0, 1, 2, 3, 4, 5, 6, 7, 8,9, or 10.

In a further embodiment m3 is 0-5, such as 0, 1, 2, 3, 4, 5 or 6.

In a further embodiment m4 is 0-5, such as 0, 1, 2, 3, 4, 5 or 6.

In a further embodiment m5 is 0-10, such as 0, 1, 2, 3, 4, 5, 6, 7, 8,9, or 10.

In a further embodiment m6 is 0-5, such as 0, 1, 2, 3, 4, 5 or 6.

In a further embodiment m7 is 0-5, such as 0, 1, 2, 3, 4, 5 or 6.

In a further embodiment n1 is 0-10, such as 0, 1, 2, 3, 4, 5 or 6.

In a further embodiment n2 is 0-10, such as 0, 1, 2, 3, 4, 5 or 6.

In a further embodiment n3 is 0-10, such as 0, 1, 2, 3, 4, 5 or 6.

In a further embodiment n4 is 0-10, such as 0, 1, 2, 3, 4, 5 or 6.

In a further embodiment X₁ is—W₁—[(CHR¹)_(I1)—W₂]_(m1)—{[(CH₂)_(n1)O]_(m2)—[(CHR²)_(I2)—W₃]_(m3)}_(n2)—and X₂ is—[(CHR₃)_(I3)—W₄]_(m4)—{[(CH₂)_(n3)O]_(m5)—[(CHR⁴)_(I4)—W₅}_(n4)—,wherein —{[(CH₂)_(n1)O]_(m2)—[(CHR²)_(Is)—W₃]_(m3)}_(n2)— and—{[(CH₂)_(n3)O]_(m5)—[(CHR⁴)_(I4)—W₅]_(m6)}_(n4)— are selected from,

wherein * is intended to denote a point of attachment, ie, an open bond.

In a further embodiment the molar weight of said hydrophilic spacer isin the range from 80 Daltons (D) to 1500 D or in the range from 500 D to1100 D.

In a still further embodiment W has the formula

—W₇—Y—,

wherein

Y is —(CH₂)₁₇—C₃₋₁₀-Cycloalkyl-W₈— or a valence bond,

I7 is 0-6,

-   W₇ is selected from —C(O)NH—, —NHC(O)—, —C(O)NHCH₂—, —CH₂NHC(O)—,    —C(O)NHS(O)₂—, —S(O)₂NHC(O)—, —OC(O)NH—, —NHC(O)O—, —C(O)CH₂—,    —CH₂C(O)—, —C(O)CH═CH—, —CH═CHC(O)—, —(CH₂)_(s3)—, —C(O)—, —C(O)O—,    —OC(O)—, or a valence bond;    wherein s3 is 0 or 1,-   W₈ is selected from —C(O)NH—, —NHC(O)—, —C(O)NHCH₂—, —CH₂NHC(O)—,    —C(O)NHS(O)₂—, —S(O)₂NHC(O)—, —OC(O)NH—, —NHC(O)O—, —C(O)CH₂—,    —CH₂C(O)—, —C(O)CH═CH—, —CH═CHC(O)—, —(CH₂)_(s4)—, —C(O)—, —C(O)O—,    —OC(O)—, or a valence bond;    wherein s4 is 0 or 1.

In an embodiment of W Y is —(CH₂)_(I7)-cyclohexyl-W₈—.

In a further embodiment Y is a valence bond.

In an embodiment W₇ is selected from —C(O)NH—, —NHC(O)—, —C(O)NHCH₂—,—CH₂NHC(O)—, —C(O)NHS(O)₂—, —S(O)₂NHC(O)— or a valence bond. Typically,W₇ is selected from —C(O)NH—, —NHC(O)—, —C(O)NHS(O)₂.

In a further embodiment W₈ is selected from —C(O)NH—, —NHC(O)—,—C(O)NHCH₂—, —CH₂NHC(O)—, —C(O)NHS(O)₂—, —S(O)₂NHC(O)— or a valencebond. Typically, W₈ is selected from —C(O)NH—, —NHC(O)—, —C(O)NHS(O)₂.

In a further embodiment I7 is 0 or 1.

In a further embodiment the hydrophilic spacer B of the presentinvention is selected from

wherein * is intended to denote a point of attachment, ie, an open bond.

The albumin binding residue (substituent A in formula (I) or (II) above)attached to the growth hormone compound of the present invention is alipophilic residue, which binds non-covalently to albumin. Typically,albumin binding residue is negatively charged at physiological pH, andhas a binding affinity towards human serum albumin that is below about10 μM or even below about 1 μM.

In a further embodiment of the growth hormone compound of the presentinvention the albumin binding residue is selected from a straight chainalkyl group, a branched alkyl group, a group which has a w-carboxylicacid group or a w-carboxylic acid isoster. Typically, the albuminbinding residue has from 6 to 40 carbon atoms. In a further embodimentthe albumin binding residue has from 8 to 26 carbon atoms. In a furtherembodiment the albumin binding residue has from 8 to 20 carbon atoms.

In a further embodiment A has 14 to 26 carbon atoms and comprises anω-carboxylic acid group. In a further embodiment A has 14 to 26 carbonatoms and comprises an ω-carboxylic acid isoster, such as a tetrazol.

In a further embodiment A is selected from

wherein * denotes the attachment to B through W.

The hydrophilic spacer (B) is preferably introduced in a position of thegrowth hormone compound (GH) in a selective manner in order to be ableto control whether one, two, or three albumin binding residues (A)should be incorporated in the growth hormone compound. The hydrophilicspacer (B) may be attached to an amino acid side-chain of the GHcompound. Such amino acid side-chain may be a chemically modified aminoacid side-chain of the GH compound. Another, such amino acid side-chainmay be an enzymatically modified amino acid side-chain of the GHcompound. Preferably, a transglutaminase is used to introduce ahydrophilic spacer in the glutamine residue in the positioncorresponding to position 40 or 141 in SEQ ID No. 1. Another way ofselectively introducing a hydrophilic spacer is in the N-terminalresidue of the growth hormone compound, such as hGH (SEQ ID No. 1).

In the growth hormone conjugate of the formula (I) the fragment A-W—Bmay be linear or branched. In one embodiment, A-W—B is not a linearpeptide.

In a further embodiment the albumin binding residue via a hydrophilicspacer is attached to the glutamine residue in the positioncorresponding to position 40 in SEQ ID No. 1.

In a further embodiment the albumin binding residue via a hydrophilicspacer is attached to the glutamine residue in the positioncorresponding to position 141 in SEQ ID No. 1.

In a further embodiment the albumin binding residue via a hydrophilicspacer is attached to the N-terminal residue of the growth hormonecompound, such as hGH (SEQ ID No. 1).

In a further embodiment the albumin binding residue via a hydrophilicspacer is attached to the glutamine residue in the positioncorresponding to position 40 and to the glutamine residue in theposition corresponding to position 141 in SEQ ID No. 1.

In a further embodiment the albumin binding residue via a hydrophilicspacer is attached to the glutamine residue in the positioncorresponding to position 40 and to the N-terminal residue of the growthhormone compound, such as hGH (SEQ ID No. 1).

In a further embodiment the albumin binding residue via a hydrophilicspacer is attached to the glutamine residue in the positioncorresponding to position 141 and to the N-terminal residue of thegrowth hormone compound, such as hGH (SEQ ID No. 1).

The growth hormone conjugates of the present invention are selected from

In a further aspect the present invention relates to a growth hormoneconjugate which comprises a growth hormone compound (GH) linked to analbumin binding residue via a hydrophilic spacer, or a pharmaceuticallyacceptable salt, solvate or prodrug thereof for use in therapy.Furthermore, in the growth hormone conjugate of the present inventionGH, the albumin binding residue, and the hydrophilic spacer are selectedfrom any one of the above embodiments, in particular the growth hormoneconjugate has the formula (I) or (II).

In a further aspect the present invention relates to a pharmaceuticalcomposition comprising a growth hormone conjugate which comprises agrowth hormone compound (GH) linked to an albumin binding residue via ahydrophilic spacer, or a pharmaceutically acceptable salt, solvate orprodrug thereof, optionally in combination with a pharmaceuticalacceptable excipient.

The term “identity” as known in the art, refers to a relationshipbetween the sequences of two or more proteins, as determined bycomparing the sequences. In the art, “identity” also means the degree ofsequence relatedness between proteins, as determined by the number ofmatches between strings of two or more amino acid residues. “Identity”measures the percent of identical matches between the smaller of two ormore sequences with gap alignments (if any) addressed by a particularmathematical model or computer program (i.e., “algorithms”). Identity ofrelated proteins can be readily calculated by known methods. Suchmethods include, but are not limited to, those described inComputational Molecular Biology, Lesk, A. M., ed., Oxford UniversityPress, New York, 1988; Biocomputing: Informatics and Genome Projects,Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis ofSequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., HumanaPress, New Jersey, 1994; Sequence Analysis in Molecular Biology, vonHeinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M.and Devereux, J., eds., M. Stockton Press, New York, 1991; and Carilloet al., SIAM J. Applied Math., 48, 1073, (1988).

Preferred methods to determine identity are designed to give the largestmatch between the sequences tested. Methods to determine identity aredescribed in publicly available computer programs. Preferred computerprogram methods to determine identity between two sequences include theGCG program package, including GAP (Devereux et al., Nucl. Acid. Res.,12, 387, (1984); Genetics Computer Group, University of Wisconsin,Madison, Wis.), BLASTP, BLASTN, and FASTA (Altschul et al., J. Mol.Biol., 215, 403-410, (1990)). The BLASTX program is publicly availablefrom the National Center for Biotechnology Information (NCBI) and othersources (BLAST Manual, Altschul et al. NCB/NLM/NIH Bethesda, Md. 20894;Altschul et al., supra). The well known Smith Waterman algorithm mayalso be used to determine identity.

For example, using the computer algorithm GAP (Genetics Computer Group,University of Wisconsin, Madison, Wis.), two proteins for which thepercent sequence identity is to be determined are aligned for optimalmatching of their respective amino acids (the “matched span”, asdetermined by the algorithm). A gap opening penalty (which is calculatedas 3.times. the average diagonal; the “average diagonal” is the averageof the diagonal of the comparison matrix being used; the “diagonal” isthe score or number assigned to each perfect amino acid match by theparticular comparison matrix) and a gap extension penalty (which isusually 1/10 times the gap opening penalty), as well as a comparisonmatrix such as PAM 250 or BLOSUM 62 are used in conjunction with thealgorithm. A standard comparison matrix (see Dayhoff et al., Atlas ofProtein Sequence and Structure, vol. 5, supp.3 (1978) for the PAM 250comparison matrix; Henikoff et al., Proc. Natl. Acad. Sci USA, 89,10915-10919, (1992) for the BLOSUM 62 comparison matrix) is also used bythe algorithm.

Preferred parameters for a protein sequence comparison include thefollowing:

Algorithm: Needleman et al., J. Mol. Biol, 48, 443-453, (1970);Comparison matrix: BLOSUM 62 from Henikoff et al., Proc. Natl. Acad.Sci. USA, 89, 10915-10919, (1992); Gap Penalty: 12, Gap Length Penalty:4, Threshold of Similarity: 0.

The GAP program is useful with the above parameters. The aforementionedparameters are the default parameters for protein comparisons (alongwith no penalty for end gaps) using the GAP algorithm.

The compounds of the present invention have improved pharmacologicalproperties compared to the corresponding un-conjugated growth hormone,which is also referred to as the parent compound. Examples of suchpharmacological properties include functional in vivo half-life,immunogencity, renal filtration, protease protection and albuminbinding.

The term “functional in vivo half-life” is used in its normal meaning,i.e., the time at which 50% of the biological activity of the GH or GHconjugate is still present in the body/target organ, or the time atwhich the activity of the GH or GH conjugate is 50% of its initialvalue. As an alternative to determining functional in vivo half-life,“in vivo plasma half-life” may be determined, i.e., the time at which50% of the GH or GH conjugate circulate in the plasma or bloodstreamprior to being cleared. Determination of plasma half-life is often moresimple than determining functional half-life and the magnitude of plasmahalf-life is usually a good indication of the magnitude of functional invivo half-life. Alternative terms to plasma half-life include serumhalf-life, circulating half-life, circulatory half-life, serumclearance, plasma clearance, and clearance half-life.

The term “increased” as used in connection with the functional in vivohalf-life or plasma half-life is used to indicate that the relevanthalf-life of the GH conjugate is statistically significantly increasedrelative to that of the parent GH, as determined under comparableconditions. For instance the relevant half-life may be increased by atleast about 25%, such as by at lest about 50%, e.g., by at least about100%, 150%, 200%, 250%, or 500%. In one embodiment, the compounds of thepresent invention exhibit an increase in half-life of at least about 5h, preferably at least about 24 h, more preferably at least about 72 h,and most preferably at least about 7 days, relative to the half-life ofthe parent GH.

Measurement of in vivo plasma half-life can be carried out in a numberof ways as described in the literature. An increase in in vivo plasmahalf-life may be quantified as a decrease in clearance (CL) or as anincrease in mean residence time (MRT). Conjugated GH of the presentinvention for which the CL is decreased to less than 70%, such as lessthan 50%, such than less than 20%, such than less than 10% of the CL ofthe parent GH as determined in a suitable assay is said to have anincreased in vivo plasma half-life. Conjugated GH of the presentinvention for which MRT is increased to more than 130%, such as morethan 150%, such as more than 200%, such as more than 500% of the MRT ofthe parent GH in a suitable assay is said to have an increased in vivoplasma half-life. Clearance and mean residence time can be assessed instandard pharmacokinetic studies using suitable test animals. It iswithin the capabilities of a person skilled in the art to choose asuitable test animal for a given protein. Tests in human, of course,represent the ultimate test. Suitable text animals include normal,Sprague-Dawley male rats, mice and cynomolgus monkeys. Typically themice and rats are in injected in a single subcutaneous bolus, whilemonkeys may be injected in a single subcutaneous bolus or in a single ivdose. The amount injected depends on the test animal. Subsequently,blood samples are taken over a period of one to five days as appropriatefor the assessment of CL and MRT. The blood samples are convenientlyanalysed by ELISA techniques.

The term “Immunogenicity” of a compound refers to the ability of thecompound, when administered to a human, to elicit a deleterious immuneresponse, whether humoral, cellular, or both. In any humansub-population, there may be individuals who exhibit sensitivity toparticular administered proteins. Immunogenicity may be measured byquantifying the presence of growth hormone antibodies and/or growthhormone responsive T-cells in a sensitive individual, using conventionalmethods known in the art. In one embodiment, the conjugated GH of thepresent invention exhibit a decrease in immunogenicity in a sensitiveindividual of at least about 10%, preferably at least about 25%, morepreferably at least about 40% and most preferably at least about 50%,relative to the immunogenicity for that individual of the parent GH.

The term “protease protection” or “protease protected” as used herein isintended to indicate that the conjugated GH of the present invention ismore resistant to the plasma peptidase or proteases than is the parentGH. Protease and peptidase enzymes present in plasma are known to beinvolved in the degradation of circulating proteins, such as e.g.circulating peptide hormones, such as growth hormone.

Growth hormone may be susceptible to degradation by for instancethrombin, plasmin, subtilisin, and chymotrypsin-like serine proteinase.Assays for determination of degradation of these proteases are describedin J. Biotech., 65, 183, (1998). In one embodiment, the rate ofhydrolysis of the GH conjugate is less than 70%, such as less than 40%,such as less than 10% of that of the parent GH.

The most abundant protein component in circulating blood of mammalianspecies is serum albumin, which is normally present at a concentrationof approximately 3 to 4.5 grams per 100 milliliters of whole blood.Serum albumin is a blood protein of approximately 70,000 daltons whichhas several important functions in the circulatory system. It functionsas a transporter of a variety of organic molecules found in the blood,as the main transporter of various metabolites such as fatty acids andbilirubin through the blood, and, owing to its abundance, as an osmoticregulator of the circulating blood. Serum albumin has a half-life ofmore than one week, and one approach to increasing the plasma half-lifeof proteins has been to conjugate to the protein a group that binds toserum albumin. Albumin binding property may be determined as describedin J. Med. Chem., 43, 1986, (2000) which is incorporated herein byreference.

The growth hormone conjugates of formula (I) or (II) exert growthhormone activity and may as such be used in the treatment of diseases orstates which will benefit from an increase in the amount of circulatinggrowth hormone. In particular, the invention provides a method for thetreatment of growth hormone deficiency (GHD); Turner Syndrome;Prader-Willi syndrome (PWS); Noonan syndrome; Down syndrome; chronicrenal disease, juvenile rheumatoid arthritis; cystic fibrosis,HIV-infection in children receiving HAART treatment (HIV/HALS children);short children born short for gestational age (SGA); short stature inchildren born with very low birth weight (VLBW) but SGA; skeletaldysplasia; hypochondroplasia; achondroplasia; idiopathic short stature(ISS); GHD in adults; fractures in or of long bones, such as tibia,fibula, femur, humerus, radius, ulna, clavicula, matacarpea, matatarsea,and digit; fractures in or of spongious bones, such as the scull, baseof hand, and base of food; patients after tendon or ligament surgery ine.g. hand, knee, or shoulder; patients having or going throughdistraction osteogenesis; patients after hip or discus replacement,meniscus repair, spinal fusions or prosthesis fixation, such as in theknee, hip, shoulder, elbow, wrist or jaw; patients into whichosteosynthesis material, such as nails, screws and plates, have beenfixed; patients with non-union or mal-union of fractures; patients afterosteatomia, e.g. from tibia or 1^(st) toe; patients after graftimplantation; articular cartilage degeneration in knee caused by traumaor arthritis; osteoporosis in patients with Turner syndrome;osteoporosis in men; adult patients in chronic dialysis (APCD);malnutritional associated cardiovascular disease in APCD; reversal ofcachexia in APCD; cancer in APCD; chronic abstractive pulmonal diseasein APCD; HIV in APCD; elderly with APCD; chronic liver disease in APCD,fatigue syndrome in APCD; Crohn's disease; impaired liver function;males with HIV infections; short bowel syndrome; central obesity;HIV-associated lipodystrophy syndrome (HALS); male infertility; patientsafter major elective surgery, alcohol/drug detoxification orneurological trauma; aging; frail elderly; osteo-arthritis;traumatically damaged cartilage; erectile dysfunction; fibromyalgia;memory disorders; depression; traumatic brain injury; subarachnoidhaemorrhage; very low birth weight; metabolic syndrome; glucocorticoidmyopathy; or short stature due to glucucorticoid treatment in children,the method comprising administering to a patient in need thereof atherapeutically effective amount of a growth hormone conjugate accordingto formula (I) or (II).

In a further aspect, the invention provides a method for theacceleration of the healing of muscle tissue, nervous tissue or wounds;the acceleration or improvement of blood flow to damaged tissue; or thedecrease of infection rate in damaged tissue, the method comprisingadministration to a patient in need thereof an effective amount of atherapeutically effective amount of a growth hormone conjugate offormula (I) or (II).

In a further embodiment, the invention relates to the use of a growthhormone conjugate of formula (I) or (II) in the manufacture of diseasesbenefiting from an increase in the growth hormone plasma level, such asthe disease mentioned above.

A typical parenteral dose is in the range of 10⁻⁹ mg/kg to about 100mg/kg body weight per administration. Typical administration doses arefrom about 0.0000001 to about 10 mg/kg body weight per administration.The exact dose will depend on e.g. indication, medicament, frequency andmode of administration, the sex, age and general condition of thesubject to be treated, the nature and the severity of the disease orcondition to be treated, the desired effect of the treatment and otherfactors evident to the person skilled in the art.

Typical dosing frequencies are twice daily, once daily, bi-daily, twiceweekly, once weekly or with even longer dosing intervals. Due to theprolonged half-lives of the fusion proteins of the present invention, adosing regime with long dosing intervals, such as twice weekly, onceweekly or with even longer dosing intervals is a particular embodimentof the invention.

Many diseases are treated using more than one medicament in thetreatment, either concomitantly administered or sequentiallyadministered. It is therefore within the scope of the present inventionto use a growth hormone conjugate of formula (I) or (II) in therapeuticmethods for the treatment of one of the above mentioned diseases incombination with one or more other therapeutically active compound(s)normally used in the treatment said diseases. By analogy, it is alsowithin the scope of the present invention to use a growth hormoneconjugate of formula (I) or (II)in combination with othertherapeutically active compounds normally used in the treatment of oneof the above mentioned diseases in the manufacture of a medicament forsaid disease.

General Methods of Preparation

Enzyme Conjugation:

In the preparation of a growth hormone conjugate of the presentinvention, typically at least one of the covalent bonds established inthe preparation of a A-W—B-GH conjugate of formula (I) is prepared byuse of an enzyme as illustrated in the examples below. Such an enzymemay for instance be selected from the group consisting oftransglutaminases, serine proteases and cysteine proteases. Typically,said enzyme is a transglutaminase. Such transglutaminase may forinstance be selected from the group consisting of microbialtransglutaminases, tissue transglutaminases and factor XIII and variantsthereof. In another embodiment, said enzyme is a cysteine protease. Sucha cysteine protease may for instance be selected from the groupconsisting of papain, sortase A and sortase B. In a further embodiment,said enzyme is a serine protease. Such a serine protease may forinstance be selected from the group consisting of carboxypeptidase Y(CPY) (PCT application WO2005/035553 contains general disclosure ofprotein modification using CPY), trypsin and chymotrypsin.

The growth hormone conjugate of the present invention may be prepared bymany different methods, non-limiting examples are shown below.

The present invention also provides methods for preparing A-W—B-GHconjugates of formula (I).

Transglutaminase

As stated above, at least one of the covalent bonds established in thepreparation of a A-W—B-GH conjugate of the present invention may beprepared by use of a transglutaminase. Transglutaminases may includemicrobial transglutaminases such as that isolated from the Streptomycesspecies; S. mobaraense, S. cinnamoneum, S. griseocarneum (U.S. Pat. No.5,156,956 incorporated herein by reference), S. lavendulae (U.S. Pat.No. 5,252,469 incorporated herein by reference) and Streptomycesladakanum (JP2003/199569 incorporated herein by reference). Other usefulmicrobial transglutaminases have been isolated from Bacillus subtilis(disclosed in U.S. Pat. No. 5,731,183, which is incorporated herein byreference) and from various Myxomycetes. Other examples of usefulmicrobial transglutaminases are those disclosed in WO96/06931 (e.g.transglutaminase from Bacillus lydicus) and WO96/22366, both of whichare incorporated herein by reference. Useful non-microbialtransglutaminases include guinea-pig liver transglutaminase, andtransglutaminases from various marine sources like the flat fish Pagrusmajor (disclosed in EP0555649, which is incorporated herein byreference), and the Japanese oyster Crassostrea gigas (disclosed in U.S.Pat. No. 5,736,356, which is incorporated herein by reference).Functional analogues and derivatives thereof may also be useful.

Typically, the TGase used in the methods of the invention is a microbialtransglutaminase. In one embodiment, the TGase is from S. mobaraense ora variant thereof, for instance as described in WO2007/020290 andWO2008/020075. In another embodiment, the TGase is from S. ladakanum ora variant thereof, for instance as described in WO2008/020075.

The conjugation of hGH to A-W—B according to the present invention maybe achieved by TGase-mediated modification leading to selectivealteration at specific lysine (Lys) or glutamine (Gln) positions in thesequence of the GH compound depending on the substrate used. Use ofamines as substrates will lead to modification of Glutamines whereas theuse of primary amides will lead to modification of Lysines. hGH (SEQ IDNo. 1) has 9 lysine residues at positions 38, 41, 70, 115, 140, 145,158, 168 and 172 and 13 glutamine residues at positions 22, 29, 40, 46,49, 68, 69, 84, 91, 122, 137, 141 and 181. Not all of these are readilyavailable for modification nor suitably for modifications as this willlead to diminished binding potency to the growth hormone binding proteinhence leading to reduced biological activity. The x-ray protein crystalstructure between hGH and its binding protein (pdb: 3HHR) reveals thatat least 4 lysines (38, 41, 168 and 172) takes part in binding to thebinding protein and potentially only one of the glutamines (Gln 46).This render the glutamines more attractive as target for selectiveintroduction of an albumin binder linker. These structuralconsiderations are further supported by findings summarised by N. Chêneet al in Reprod. Nutr. Develop. 29, 1-25 (1989) where it's concludedthat chemical modifications affecting lysines have been found to have anegative effect on the in vivo biological activity and on the bindingcapacity to the liver receptors of GH.

Chemistry I

In an aspect the present invention relates to preparation of a growthhormone conjugate of formula (VI) wherein a GH compound is treated witha property-modifying group-derived aldehyde or ketone to yield an amine,imine or a hemiaminal.

In a further aspect the present invention relates to preparation of agrowth hormone conjugate of formula (VI) comprising treatment of analdehyde or ketone derived from the GH compound with aproperty-modifying group-derived aniline or heteroarylamine to yield anamine, imine or a hemiaminal.

In an embodiment, aldehyde derived from the GH compound (IV) is treatedwith property-modifying group-derived aniline or heteroarylamine (V).

The term “GH compound derived aldehyde (or ketone)” or “an aldehyde orketone derived from the GH compound” is intended to indicate a GHcompound to which an aldehyde or ketone functional group has beencovalently attached, or a GH compound on which an aldehyde or ketonefunctional group has been generated. The preparation of GHcompound-derived aldehydes, such as compound (IV) illustrated below iswell known to those skilled in the art, and any of these knownprocedures may be used to prepare the GH compound-derived aldehyde (IV)required for the realization of the invention disclosed herein.

In one embodiment, the conjugate A-W—B-hGH (VI) is prepared asillustrated below:

The TGase-mediated enzymatic reaction with GH (I) results in themodification of Gln at position 141 and/or 40 affording GH (II).Conjugation of periodate oxidised GH (II) with A-W—B-NH2 (V) occurs viareductive alkylation. Reductive alkylation is exemplified herein and iswell-recognized in the art and results in GH compounds (VI) modified inposition Gln(141) and/or 40.

Chemistry II

In one embodiment, the conjugate A-W—B-hGH is prepared as illustratedbelow:

The derivatization process as shown above provides an albumin bindinglinker attached to hGH compound in position Phe(1).

A close relationship to the natural peptide is generally regarded as anadvantage with therapeutic interventions comprising administration ofvariants or analogues of this natural peptide as it minimizes the riskof e.g. any unwanted antibody generation.

Many nucleophilic compounds are known which could be incorporated intopeptides according to the methods of the present invention, and a-aminoacids are one such type of nucleophilic compounds. For the purpose ofthe present invention, it is, however, preferred to select thenucleophilic compound so that the transacylated compound formed is notitself a substrate for the enzyme applied. Stated differently, it ispreferred to apply a nucleophilic compound which effectively blocks anyfurther reaction of the enzyme. One example of such compounds is amidesof a-amino acids as carboxy amidated peptides are not substrates forcarboxypeptidases.

It is recognised that whether or not a compound is a substrate for agiven enzyme in principle depends on the conditions, e.g. the timeframe, under which the reaction takes place. Given sufficient time, manycompounds are, in fact, substrates for an enzyme although they are notunder normal conditions regarded as such. When it is stated above thatthe transacylated compound itself should not be a substrate of theenzyme it is intended to indicate that the transacylated compound itselfis not a substrate for the enzyme to an extent where the followingreactions in the method of the present invention are disturbed. If thetransacylated compound is, in fact, a substrate for the enzyme, theenzyme may be removed or inactivated, e.g. by enzyme inhibitors,following the transacylation reaction.

Pharmaceutical Compositions

Another purpose is to provide a pharmaceutical composition comprising agrowth hormone conjugate of the present invention, such as a growthhormone conjugate of formula (I) or (II), which is present in aconcentration from 10⁻¹⁵ mg/mL to 200 mg/mL, such as e.g. 10⁻¹⁰ mg/mL to5 mg/mL and wherein said composition has a pH from 2.0 to 10.0. Thecomposition may further comprise pharmaceutical exhibits, such as abuffer system, preservative(s), tonicity agent(s), chelating agent(s),stabilizers and surfactants. In one embodiment of the invention thepharmaceutical composition is an aqueous composition, i.e. compositioncomprising water. Such composition is typically a solution or asuspension. In a further embodiment of the invention the pharmaceuticalcomposition is an aqueous solution. The term “aqueous composition” isdefined as a composition comprising at least 50% w/w water. Likewise,the term “aqueous solution” is defined as a solution comprising at least50% w/w water, and the term “aqueous suspension” is defined as asuspension comprising at least 50% w/w water.

In another embodiment the pharmaceutical composition is a freeze-driedcomposition, whereto the physician or the patient adds solvents and/ordiluents prior to use.

In another embodiment the pharmaceutical composition is a driedcomposition (e.g. freeze-dried or spray-dried) ready for use without anyprior dissolution.

In a further aspect the invention relates to a pharmaceuticalcomposition comprising an aqueous solution of a growth hormoneconjugate, such as a growth hormone conjugate of formula (I) or (II),and a buffer, wherein said GH conjugate is present in a concentrationfrom 0.1-100 mg/mL or above, and wherein said composition has a pH fromabout 2.0 to about 10.0.

In a another embodiment of the invention the pH of the composition isselected from the list consisting of 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6,2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0,4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4,5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8,6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2,8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6,9.7, 9.8, 9.9, and 10.0.

In a further embodiment of the invention the buffer is selected from thegroup consisting of sodium acetate, sodium carbonate, citrate,glycylglycine, histidine, glycine, lysine, arginine, sodium dihydrogenphosphate, disodium hydrogen phosphate, sodium phosphate, andtris(hydroxymethyl)-aminomethan, bicine, tricine, malic acid, succinate,maleic acid, fumaric acid, tartaric acid, aspartic acid or mixturesthereof. Each one of these specific buffers constitutes an alternativeembodiment of the invention.

In a further embodiment of the invention the composition furthercomprises a pharmaceutically acceptable preservative. In a furtherembodiment of the invention the preservative is selected from the groupconsisting of phenol, o-cresol, m-cresol, p-cresol, methylp-hydroxybenzoate, propyl p-hydroxybenzoate, 2-phenoxyethanol, butylp-hydroxybenzoate, 2-phenylethanol, benzyl alcohol, chlorobutanol, andthiomerosal, bronopol, benzoic acid, imidurea, chlorohexidine, sodiumdehydroacetate, chlorocresol, ethyl p-hydroxybenzoate, benzethoniumchloride, chlorphenesine (3p-chlorphenoxypropane-1,2-diol) or mixturesthereof. In a further embodiment of the invention the preservative ispresent in a concentration from 0.1 mg/mL to 20 mg/mL. In a furtherembodiment of the invention the preservative is present in aconcentration from 0.1 mg/mL to 5 mg/mL. In a further embodiment of theinvention the preservative is present in a concentration from 5 mg/mL to10 mg/mL. In a further embodiment of the invention the preservative ispresent in a concentration from 10 mg/mL to 20 mg/mL. Each one of thesespecific preservatives constitutes an alternative embodiment of theinvention. The use of a preservative in pharmaceutical compositions iswell-known to the skilled person. For convenience reference is made toRemington: The Science and Practice of Pharmacy, 20^(th) edition, 2000.

In a further embodiment of the invention the composition furthercomprises an isotonic agent. In a further embodiment of the inventionthe isotonic agent is selected from the group consisting of a salt (e.g.sodium chloride), a sugar or sugar alcohol, an amino acid (e.g.L-glycine, L-histidine, arginine, lysine, isoleucine, aspartic acid,tryptophan, threonine), an alditol (e.g. glycerol (glycerine),1,2-propanediol (propyleneglycol), 1,3-propanediol, 1,3-butanediol)polyethyleneglycol (e.g. PEG 400), or mixtures thereof. Any sugar suchas mono-, di-, or polysaccharides, or water-soluble glucans, includingfor example fructose, glucose, mannose, sorbose, xylose, maltose,lactose, sucrose, trehalose, dextran, pullulan, dextrin, cyclodextrin,soluble starch, hydroxyethyl starch and carboxymethylcellulose-Na may beused. In one embodiment the sugar additive is sucrose. Sugar alcohol isdefined as a C4-C8 hydrocarbon having at least one —OH group andincludes, for example, mannitol, sorbitol, inositol, galactitol,dulcitol, xylitol, and arabitol. In one embodiment the sugar alcoholadditive is mannitol. The sugars or sugar alcohols mentioned above maybe used individually or in combination. There is no fixed limit to theamount used, as long as the sugar or sugar alcohol is soluble in theliquid preparation and does not adversely effect the stabilizing effectsobtained using the methods of the invention. In one embodiment, thesugar or sugar alcohol concentration is between about 1 mg/mL and about150 mg/mL. In a further embodiment of the invention the isotonic agentis present in a concentration from 1 mg/mL to 50 mg/mL. In a furtherembodiment of the invention the isotonic agent is present in aconcentration from 1 mg/mL to 7 mg/mL. In a further embodiment of theinvention the isotonic agent is present in a concentration from 8 mg/mLto 24 mg/mL. In a further embodiment of the invention the isotonic agentis present in a concentration from 25 mg/mL to 50 mg/mL. Each one ofthese specific isotonic agents constitutes an alternative embodiment ofthe invention. The use of an isotonic agent in pharmaceuticalcompositions is well-known to the skilled person. For conveniencereference is made to Remington: The Science and Practice of Pharmacy,20^(th) edition, 2000.

In a further embodiment of the invention the composition furthercomprises a chelating agent. In a further embodiment of the inventionthe chelating agent is selected from salts of ethylenediaminetetraaceticacid (EDTA), citric acid, and aspartic acid, and mixtures thereof. In afurther embodiment of the invention the chelating agent is present in aconcentration from 0.1 mg/mL to 5 mg/mL. In a further embodiment of theinvention the chelating agent is present in a concentration from 0.1mg/mL to 2 mg/mL. In a further embodiment of the invention the chelatingagent is present in a concentration from 2 mg/mL to 5 mg/mL. Each one ofthese specific chelating agents constitutes an alternative embodiment ofthe invention. The use of a chelating agent in pharmaceuticalcompositions is well-known to the skilled person. For conveniencereference is made to Remington: The Science and Practice of Pharmacy,20^(th) edition, 2000.

In a further embodiment of the invention the composition furthercomprises a stabilizer. The use of a stabilizer in pharmaceuticalcompositions is well-known to the skilled person. For conveniencereference is made to Remington: The Science and Practice of Pharmacy,20^(th) edition, 2000.

More particularly, compositions of the invention are stabilized liquidpharmaceutical compositions whose therapeutically active componentsinclude a protein that possibly exhibits aggregate formation duringstorage in liquid pharmaceutical compositions. By “aggregate formation”is intended a physical interaction between the protein molecules thatresults in formation of oligomers, which may remain soluble, or largevisible aggregates that precipitate from the solution. By “duringstorage” is intended a liquid pharmaceutical composition or compositiononce prepared, is not immediately administered to a subject. Rather,following preparation, it is packaged for storage, either in a liquidform, in a frozen state, or in a dried form for later reconstitutioninto a liquid form or other form suitable for administration to asubject. By “dried form” is intended the liquid pharmaceuticalcomposition or composition is dried either by freeze drying (i.e.,lyophilization; see, for example, Williams and Polli, J. Parenteral Sci.Technol. 38, 48-59, (1984)), spray drying (see Masters (1991) inSpray-Drying Handbook (5^(th) ed; Longman Scientific and Technical,Essez, U.K.), pp. 491-676; Broadhead et al. (1992) Drug Devel. Ind.Pharm. 18, 1169-1206, (1992); and Mumenthaler et al., Pharm. Res. 11,12-20 (1994)), or air drying (Carpenter and Crowe, Cryobiology 25,459-470, (1988); and Roser, Biopharm. 4, 47-53, (1991)). Aggregateformation by a protein during storage of a liquid pharmaceuticalcomposition can adversely affect biological activity of that protein,resulting in loss of therapeutic efficacy of the pharmaceuticalcomposition. Furthermore, aggregate formation may cause other problemssuch as blockage of tubing, membranes, or pumps when theprotein-containing pharmaceutical composition is administered using aninfusion system.

The pharmaceutical compositions of the invention may further comprise anamount of an amino acid base sufficient to decrease aggregate formationby the protein during storage of the composition. By “amino acid base”is intended an amino acid or a combination of amino acids, where anygiven amino acid is present either in its free base form or in its saltform. Where a combination of amino acids is used, all of the amino acidsmay be present in their free base forms, all may be present in theirsalt forms, or some may be present in their free base forms while othersare present in their salt forms. In one embodiment, amino acids to usein preparing the compositions of the invention are those carrying acharged side chain, such as arginine, lysine, aspartic acid, andglutamic acid. Any stereoisomer (i.e., L or D isomer, or mixturesthereof) of a particular amino acid (methionine, histidine, arginine,lysine, isoleucine, aspartic acid, tryptophan, threonine and mixturesthereof) or combinations of these stereoisomers or glycine or an organicbase such as but not limited to imidazole, may be present in thepharmaceutical compositions of the invention so long as the particularamino acid or organic base is present either in its free base form orits salt form. In one embodiment the L-stereoisomer of an amino acid isused. In one embodiment the L-stereoisomer is used. Compositions of theinvention may also be formulated with analogues of these amino acids. By“amino acid analogue” is intended a derivative of the naturallyoccurring amino acid that brings about the desired effect of decreasingaggregate formation by the protein during storage of the liquidpharmaceutical compositions of the invention. Suitable arginineanalogues include, for example, aminoguanidine, ornithine andN-monoethyl L-arginine, suitable methionine analogues include ethionineand buthionine and suitable cysteine analogues include S-methyl-Lcysteine. As with the other amino acids, the amino acid analogues areincorporated into the compositions in either their free base form ortheir salt form. In a further embodiment of the invention the aminoacids or amino acid analogues are used in a concentration, which issufficient to prevent or delay aggregation of the protein.

In a further embodiment of the invention methionine (or other sulphuricamino acids or amino acid analogous) may be added to inhibit oxidationof methionine residues to methionine sulfoxide when the protein actingas the therapeutic agent is a protein comprising at least one methionineresidue susceptible to such oxidation. By “inhibit” is intended minimalaccumulation of methionine oxidized species over time. Inhibitingmethionine oxidation results in greater retention of the protein in itsproper molecular form. Any stereoisomer of methionine (L or D isomer) orany combinations thereof can be used. The amount to be added should bean amount sufficient to inhibit oxidation of the methionine residuessuch that the amount of methionine sulfoxide is acceptable to regulatoryagencies. Typically, this means that the composition contains no morethan about 10% to about 30% methionine sulfoxide. Generally, this can beobtained by adding methionine such that the ratio of methionine added tomethionine residues ranges from about 1:1 to about 1000:1, such as 10:1to about 100:1.

In a further embodiment of the invention the composition furthercomprises a stabilizer selected from the group of high molecular weightpolymers or low molecular compounds. In a further embodiment of theinvention the stabilizer is selected from polyethylene glycol (e.g. PEG3350), polyvinyl alcohol (PVA), polyvinylpyrrolidone,carboxy-/hydroxycellulose or derivatives thereof (e.g. HPC, HPC-SL,HPC-L and HPMC), cyclodextrins, sulphur-containing substances asmonothioglycerol, thioglycolic acid and 2-methylthioethanol, anddifferent salts (e.g. sodium chloride). Each one of these specificstabilizers constitutes an alternative embodiment of the invention.

The pharmaceutical compositions may also comprise additional stabilizingagents, which further enhance stability of a therapeutically activeprotein therein. Stabilizing agents of particular interest to thepresent invention include, but are not limited to, methionine and EDTA,which protect the protein against methionine oxidation, and a nonionicsurfactant, which protects the protein against aggregation associatedwith freeze-thawing or mechanical shearing.

In a further embodiment of the invention the composition furthercomprises a surfactant. In a further embodiment of the invention thesurfactant is selected from a detergent, ethoxylated castor oil,polyglycolyzed glycerides, acetylated monoglycerides, sorbitan fattyacid esters, polyoxypropylene-polyoxyethylene block polymers (eg.poloxamers such as Pluronic® F68, poloxamer 188 and 407, Triton X-100),polyoxyethylene sorbitan fatty acid esters, polyoxyethylene andpolyethylene derivatives such as alkylated and alkoxylated derivatives(tweens, e.g. Tween-20, Tween-40, Tween-80 and Brij-35), monoglyceridesor ethoxylated derivatives thereof, diglycerides or polyoxyethylenederivatives thereof, alcohols, glycerol, lectins and phospholipids (eg.phosphatidyl serine, phosphatidyl choline, phosphatidyl ethanolamine,phosphatidyl inositol, diphosphatidyl glycerol and sphingomyelin),derivatives of phospholipids (eg. dipalmitoyl phosphatidic acid) andlysophospholipids (eg. palmitoyl lysophosphatidyl-L-serine and1-acyl-sn-glycero-3-phosphate esters of ethanolamine, choline, serine orthreonine) and alkyl, alkoxyl (alkyl ester), alkoxy (alkylether)-derivatives of lysophosphatidyl and phosphatidylcholines, e.g.lauroyl and myristoyl derivatives of lysophosphatidylcholine,dipalmitoylphosphatidylcholine, and modifications of the polar headgroup, that is cholines, ethanolamines, phosphatidic acid, serines,threonines, glycerol, inositol, and the positively charged DODAC, DOTMA,DCP, BISHOP, lysophosphatidylserine and lysophosphatidylthreonine, andglycerophospholipids (eg. cephalins), glyceroglycolipids (eg.galactopyransoide), sphingoglycolipids (eg. ceramides, gangliosides),dodecylphosphocholine, hen egg lysolecithin, fusidic acidderivatives—(e.g. sodium tauro-dihydrofusidate etc.), long-chain fattyacids and salts thereof C₆-C₁₂ (eg. oleic acid and caprylic acid),acylcarnitines and derivatives, N^(α)-acylated derivatives of lysine,arginine or histidine, or side-chain acylated derivatives of lysine orarginine, N^(α)-acylated derivatives of dipeptides comprising anycombination of lysine, arginine or histidine and a neutral or acidicamino acid, N^(α)-acylated derivative of a tripeptide comprising anycombination of a neutral amino acid and two charged amino acids, DSS(docusate sodium, CAS registry no [577-11-7]), docusate calcium, CASregistry no [128-49-4]), docusate potassium, CAS registry no[7491-09-0]), SDS (sodium dodecyl sulphate or sodium lauryl sulphate),sodium caprylate, cholic acid or derivatives thereof, bile acids andsalts thereof and glycine or taurine conjugates, ursodeoxycholic acid,sodium cholate, sodium deoxycholate, sodium taurocholate, sodiumglycocholate, N-Hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate,anionic (alkyl-aryl-sulphonates) monovalent surfactants, zwitterionicsurfactants (e.g. N-alkyl-N,N-dimethylammonio-1-propanesulfonates,3-cholamido-1-propyldimethylammonio-1-propanesulfonate, cationicsurfactants (quaternary ammonium bases) (e.g. cetyl-trimethylammoniumbromide, cetylpyridinium chloride), non-ionic surfactants (eg. Dodecylβ-D-glucopyranoside), poloxamines (eg. Tetronic's), which aretetrafunctional block copolymers derived from sequential addition ofpropylene oxide and ethylene oxide to ethylenediamine, or the surfactantmay be selected from the group of imidazoline derivatives, or mixturesthereof. Each one of these specific surfactants constitutes analternative embodiment of the invention.

The use of a surfactant in pharmaceutical compositions is well-known tothe skilled person. For convenience reference is made to Remington: TheScience and Practice of Pharmacy, 20^(th) edition, 2000.

It is possible that other ingredients may be present in thepharmaceutical composition of the present invention. Such additionalingredients may include wetting agents, emulsifiers, antioxidants,bulking agents, tonicity modifiers, chelating agents, metal ions,oleaginous vehicles, proteins (e.g., human serum albumin, gelatine orproteins) and a zwitterion (e.g., an amino acid such as betaine,taurine, arginine, glycine, lysine and histidine). Such additionalingredients, of course, should not adversely affect the overallstability of the pharmaceutical composition of the present invention.

Pharmaceutical compositions containing a growth hormone conjugateaccording to the present invention may be administered to a patient inneed of such treatment at several sites, for example, at topical sites,for example, skin and mucosal sites, at sites which bypass absorption,for example, administration in an artery, in a vein, in the heart, andat sites which involve absorption, for example, administration in theskin, under the skin, in a muscle or in the abdomen.

Administration of pharmaceutical compositions according to the inventionmay be through several routes of administration, for example, lingual,sublingual, buccal, in the mouth, oral, in the stomach and intestine,nasal, pulmonary, for example, through the bronchioles and alveoli or acombination thereof, epidermal, dermal, transdermal, vaginal, rectal,ocular, for examples through the conjunctiva, urethral, and parenteralto patients in need of such a treatment.

Compositions of the current invention may be administered in severaldosage forms, for example, as solutions, suspensions, emulsions,microemulsions, multiple emulsion, foams, salves, pastes, plasters,ointments, tablets, coated tablets, rinses, capsules, for example, hardgelatine capsules and soft gelatine capsules, suppositories, rectalcapsules, drops, gels, sprays, powder, aerosols, inhalants, eye drops,ophthalmic ointments, ophthalmic rinses, vaginal pessaries, vaginalrings, vaginal ointments, injection solution, in situ transformingsolutions, for example in situ gelling, in situ setting, in situprecipitating, in situ crystallization, infusion solution, and implants.

Compositions of the invention may further be compounded in, or attachedto, for example through covalent, hydrophobic and electrostaticinteractions, a drug carrier, drug-delivery system and advanced drugdelivery system in order to further enhance stability of the growthhormone conjugate, increase bioavailability, increase solubility,decrease adverse effects, achieve chronotherapy well known to thoseskilled in the art, and increase patient compliance or any combinationthereof. Examples of carriers, drug delivery systems and advanced drugdelivery systems include, but are not limited to, polymers, for examplecellulose and derivatives, polysaccharides, for example dextran andderivatives, starch and derivatives, poly(vinyl alcohol), acrylate andmethacrylate polymers, polylactic and polyglycolic acid and blockco-polymers thereof, polyethylene glycols, carrier proteins, for examplealbumin, gels, for example, thermogelling systems, for example blockco-polymeric systems well known to those skilled in the art, micelles,liposomes, microspheres, nanoparticulates, liquid crystals anddispersions thereof, L2 phase and dispersions there of, well known tothose skilled in the art of phase behaviour in lipid-water systems,polymeric micelles, multiple emulsions, self-emulsifying,self-microemulsifying, cyclodextrins and derivatives thereof, anddendrimers.

Compositions of the current invention are useful in the composition ofsolids, semi-solids, powder and solutions for pulmonary administrationof growth hormone conjugate, using, for example a metered dose inhaler,dry powder inhaler and a nebulizer, all being devices well known tothose skilled in the art.

Compositions of the current invention are specifically useful in thecomposition of controlled, sustained, protracting, retarded, and slowrelease drug delivery systems. More specifically, but not limited to,compositions are useful in composition of parenteral controlled releaseand sustained release systems (both systems leading to a many-foldreduction in number of administrations), well known to those skilled inthe art. Even more preferably, are controlled release and sustainedrelease systems administered subcutaneous. Without limiting the scope ofthe invention, examples of useful controlled release system andcompositions are hydrogels, oleaginous gels, liquid crystals, polymericmicelles, microspheres, nanoparticles,

Methods to produce controlled release systems useful for compositions ofthe current invention include, but are not limited to, crystallization,condensation, co-crystallization, precipitation, co-precipitation,emulsification, dispersion, high pressure homogenisation, encapsulation,spray drying, microencapsulating, coacervation, phase separation,solvent evaporation to produce microspheres, extrusion and supercriticalfluid processes. General reference is made to Handbook of PharmaceuticalControlled Release (Wise, D. L., ed. Marcel Dekker, New York, 2000) andDrug and the Pharmaceutical Sciences vol. 99: Protein Composition andDelivery (MacNally, E. J., ed. Marcel Dekker, New York, 2000).

Parenteral administration may be performed by subcutaneous,intramuscular, intraperitoneal or intravenous injection by means of asyringe, optionally a pen-like syringe. Alternatively, parenteraladministration can be performed by means of an infusion pump. A furtheroption is a composition which may be a solution or suspension for theadministration of the growth hormone conjugate in the form of a nasal orpulmonal spray. As a still further option, the pharmaceuticalcompositions containing the growth hormone conjugate of the inventioncan also be adapted to transdermal administration, e.g. by needle-freeinjection or from a patch, optionally an iontophoretic patch, ortransmucosal, e.g. buccal, administration.

The term “stabilized composition” refers to a composition with increasedphysical stability, increased chemical stability or increased physicaland chemical stability.

The term “physical stability” of the protein composition as used hereinrefers to the tendency of the protein to form biologically inactiveand/or insoluble aggregates of the protein as a result of exposure ofthe protein to thermo-mechanical stresses and/or interaction withinterfaces and surfaces that are destabilizing, such as hydrophobicsurfaces and interfaces. Physical stability of the aqueous proteincompositions is evaluated by means of visual inspection and/or turbiditymeasurements after exposing the composition filled in suitablecontainers (e.g. cartridges or vials) to mechanical/physical stress(e.g. agitation) at different temperatures for various time periods.Visual inspection of the compositions is performed in a sharp focusedlight with a dark background. The turbidity of the composition ischaracterized by a visual score ranking the degree of turbidity forinstance on a scale from 0 to 3 (a composition showing no turbiditycorresponds to a visual score 0, and a composition showing visualturbidity in daylight corresponds to visual score 3). A composition isclassified physical unstable with respect to protein aggregation, whenit shows visual turbidity in daylight. Alternatively, the turbidity ofthe composition can be evaluated by simple turbidity measurementswell-known to the skilled person. Physical stability of the aqueousprotein compositions can also be evaluated by using a spectroscopicagent or probe of the conformational status of the protein. The probe ispreferably a small molecule that preferentially binds to a non-nativeconformer of the protein. One example of a small molecular spectroscopicprobe of protein structure is Thioflavin T. Thioflavin T is afluorescent dye that has been widely used for the detection of amyloidfibrils. In the presence of fibrils, and perhaps other proteinconfigurations as well, Thioflavin T gives rise to a new excitationmaximum at about 450 nm and enhanced emission at about 482 nm when boundto a fibril protein form. Unbound Thioflavin T is essentiallynon-fluorescent at the wavelengths.

Other small molecules can be used as probes of the changes in proteinstructure from native to non-native states. For instance the“hydrophobic patch” probes that bind preferentially to exposedhydrophobic patches of a protein. The hydrophobic patches are generallyburied within the tertiary structure of a protein in its native state,but become exposed as a protein begins to unfold or denature. Examplesof these small molecular, spectroscopic probes are aromatic, hydrophobicdyes, such as anthracene, acridine, phenanthroline or the like. Otherspectroscopic probes are metal-amino acid complexes, such as cobaltmetal complexes of hydrophobic amino acids, such as phenylalanine,leucine, isoleucine, methionine, and valine, or the like.

The term “chemical stability” of the protein composition as used hereinrefers to chemical covalent changes in the protein structure leading toformation of chemical degradation products with potential lessbiological potency and/or potential increased immunogenic propertiescompared to the native protein structure. Various chemical degradationproducts can be formed depending on the type and nature of the nativeprotein and the environment to which the protein is exposed. Eliminationof chemical degradation can most probably not be completely avoided andincreasing amounts of chemical degradation products is often seen duringstorage and use of the protein composition as well-known by the personskilled in the art. Most proteins are prone to deamidation, a process inwhich the side chain amide group in glutaminyl or asparaginyl residuesis hydrolysed to form a free carboxylic acid. Other degradationspathways involves formation of high molecular weight transformationproducts where two or more protein molecules are covalently bound toeach other through transamidation and/or disulfide interactions leadingto formation of covalently bound dimer, oligomer and polymer degradationproducts (Stability of Protein Pharmaceuticals, Ahern. T. J. & ManningM. C., Plenum Press, New York 1992). Oxidation (of for instancemethionine residues) can be mentioned as another variant of chemicaldegradation. The chemical stability of the protein composition can beevaluated by measuring the amount of the chemical degradation productsat various time-points after exposure to different environmentalconditions (the formation of degradation products can often beaccelerated by for instance increasing temperature). The amount of eachindividual degradation product is often determined by separation of thedegradation products depending on molecule size and/or charge usingvarious chromatography techniques (e.g. SEC-HPLC and/or RP-HPLC).

Hence, as outlined above, a “stabilized composition” refers to acomposition with increased physical stability, increased chemicalstability or increased physical and chemical stability. In general, acomposition must be stable during use and storage (in compliance withrecommended use and storage conditions) until the expiration date isreached.

In one embodiment of the invention the pharmaceutical compositioncomprising the growth hormone conjugate of formula (I) or (II)is stablefor more than 6 weeks of usage and for more than 3 years of storage.

In another embodiment of the invention the pharmaceutical compositioncomprising the growth hormone conjugate of formula (I) or (II)is stablefor more than 4 weeks of usage and for more than 3 years of storage.

In a further embodiment of the invention the pharmaceutical compositioncomprising the growth hormone conjugate of formula (I) or (II) is stablefor more than 4 weeks of usage and for more than two years of storage.

In an even further embodiment of the invention the pharmaceuticalcomposition comprising the growth hormone conjugate of formula (I) or(II) is stable for more than 2 weeks of usage and for more than twoyears of storage.

All references, including publications, patent applications and patents,cited herein are hereby incorporated by reference to the same extent asif each reference was individually and specifically indicated to beincorporated by reference and was set forth in its entirety herein.

All headings and sub-headings are used herein for convenience only andshould not be construed as limiting the invention in any way,

Any combination of the above-described elements in all possiblevariations thereof is encompassed by the invention unless otherwiseindicated herein or otherwise clearly contradicted by context.

The terms “a” and “an” and “the” and similar referents as used in thecontext of describing the invention are to be construed to cover boththe singular and the plural, unless otherwise indicated herein orclearly contradicted by context.

Recitation of ranges of values herein are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range, unless otherwise indicated herein, and eachseparate value is incorporated into the specification as if it wereindividually recited herein. Unless otherwise stated, all exact valuesprovided herein are representative of corresponding approximate values(e.g., all exact exemplary values provided with respect to a particularfactor or measurement can be considered to also pro-vide a correspondingapproximate measurement, modified by “about,” where appropriate).

All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext.

The use of any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise indicated. No language in the specification should beconstrued as indicating any element is essential to the practice of theinvention unless as much is explicitly stated.

The citation and incorporation of patent documents herein is done forconvenience only and does not reflect any view of the validity,patentability and/or enforceability of such patent documents.

The invention is further described by, but not limited to, theembodiments listed here below.

-   1. A growth hormone conjugate which comprises a growth hormone    compound (GH) linked to an albumin binding residue via a hydrophilic    spacer, or a pharmaceutically acceptable salt, solvate or prodrug    thereof.-   2. The conjugate of embodiment 1 wherein the hydrophilic spacer has    mLogP<0 or a cLogP<0.5.-   3. The conjugate of embodiment 1 or 2 wherein the growth hormone    compound (GH) is linked to one albumin binding residue via a    hydrophilic spacer.-   4. The conjugate of embodiment 1 or 2 wherein the growth hormone    compound (GH) is linked to two or three albumin binding residues via    a hydrophilic spacer.-   5. The conjugate of any one of embodiments 1-4 wherein the    hydrophilic spacer has the formula

—X₁—X₂—X₃—X₄—

wherein

-   -   X₁ is        —W₁—[(CHR¹)_(I1)—W₂]_(m-1)—{[(CH₂)_(n1)E1]_(m2)-[(CHR²)_(I2)—W₃]_(m3)}_(n2)—,    -   X₂ is        —[(CHR³)_(I3)—W₄]_(m4)—{[(CH₂)_(n3)E2]_(m5)-[(CHR⁴)_(I4)—W₅]_(m6)}_(n4)—,    -   X₃ is —[(CHR⁵)₁₅—W₆]_(m7)—,    -   X₄ is —F-D1—(CH₂)_(I6)-D2-,    -   I1, I2, I3, I4, I5 and I6 independently are selected from 0-16,    -   m1, m3, m4, m6 and m7 independently are selected from 0-10,    -   m2 and m5 independently are selected from 0-25,    -   n1, n2, n3 and n4 independently are selected from 0-16,    -   F is aryl, hetaryl, pyrrolidine-2,5-dione or a valence bond,        wherein the aryl and hetaryl groups are optionally substituted        with halogen, —CN, —OH, —C(O)OH, —C(O)NH₂, —S(O)₂OH or        C₁₋₆-alkyl,    -   R¹, R², R³, R⁴ and R⁵ independently are selected from hydrogen,        —C(O)OH, —C(O)NH₂, —S(O)OH, —S(O)₂OH, —NH—C(═NH)—NH₂,        C₁₋₆-alkyl, aryl or hetaryl; wherein the alkyl, aryl and hetaryl        groups optionally are substituted with halogen, —C(O)OH,        —C(O)NH₂, —S(O)OH, —S(O)₂OH, —CN, or —OH,    -   D1, D2, E1 and E2 independently are selected from —O—, —NR⁶—,        —N(COR⁷)— or a valence bond; wherein R⁶ and R⁷ independently        represent hydrogen or C₁₋₆-alkyl,    -   W₁ to W₆ independently are selected from —C(O)NH—, —NHC(O)—,        —C(O)NHCH₂—, —CH₂NHC(O)—, —C(O)NHS(O)₂—, —S(O)₂NHC(O)—,        —OC(O)NH—, —NHC(O)O—, —C(O)CH₂—, —CH₂C(O)—, —C(O)CH═CH—,        —CH═CHC(O)—, —(CH₂)_(s1)—, —C(O)—, —C(O)O—, —OC(O)—, or a        valence bond; wherein s1 is 0 or 1.        or a pharmaceutically acceptable salt, solvate or prodrug        thereof.

-   6. A growth hormone conjugate of the formula (I):

A-W—B-GH   (I)

Wherein

-   GH represents a growth hormone compound,-   B represents a hydrophilic spacer,-   W is a chemical group linking A and B, and-   A represent an albumin binding residue; and    pharmaceutically acceptable salts, solvates and prodrugs thereof.-   7. The conjugate of embodiment 6 wherein GH represents a growth    hormone compound comprising an amino acid sequence having at least    90% identity to the amino acid sequence of human growth hormone    (hGH) (SEQ ID NO:1).-   8. The conjugate of embodiment 6 or 7, wherein GH is hGH (SEQ ID    NO:1).-   9. The conjugate of any one of embodiments 6-8 wherein B has the    formula

—X₁—X₂—X₃—X₄—

wherein

-   -   X₁ is        —W₁—[(CHR¹)_(I1)—W₂]_(m-1)—{[(CH₂)_(n1)E1]_(m2)-[(CHR²)_(I2)—W₃]_(m3)}_(n2)—,    -   X₂ is        —[(CHR³)_(I3)—W₄]_(m4)—{[(CH₂)_(n3)E2]_(m5)-[(CHR⁴)_(I4)—W₅]_(m6)}_(n4)—,    -   X₃ is —[(CHR⁵)₁₅—W₆]_(m7)—,    -   X₄ is —F-D1—(CH₂)_(I6)-D2-,    -   I1, I2, I3, I4, I5 and I6 independently are selected from 0-16,    -   m1, m3, m4, m6 and m7 independently are selected from 0-10,    -   m2 and m5 independently are selected from 0-25,

1n1, n2, n3 and n4 independently are selected from 0-16,

-   -   F is aryl, hetaryl, pyrrolidine-2,5-dione or a valence bond,        wherein the aryl and hetaryl groups are optionally substituted        with halogen, —CN, —OH, —C(O)OH, —C(O)NH₂, —S(O)₂OH or        C₁₋₆-alkyl,    -   R¹, R², R³, R⁴ and R⁵ independently are selected from hydrogen,        —C(O)OH, —C(O)NH₂, —S(O)OH, —S(O)₂OH, —NH—C(═NH)—NH₂,        C₁₋₆-alkyl, aryl or hetaryl; wherein the alkyl, aryl and hetaryl        groups optionally are substituted with halogen, —C(O)OH,        —C(O)NH₂, —S(O)OH, —S(O)₂OH, —CN, or —OH,    -   D1, D2, E1 and E2 independently are selected from —O—, —NR⁶—,        —N(COR⁷)— or a valence bond; wherein R⁶ and R⁷ independently        represent hydrogen or C₁₋₆-alkyl,    -   W₁ to W₆ independently are selected from —C(O)NH—, —NHC(O)—,        —C(O)NHCH₂—, —CH₂NHC(O)—, —C(O)NHS(O)₂—, —S(O)₂NHC(O)—,        —OC(O)NH—, —NHC(O)O—, —C(O)CH₂—, —CH₂C(O)—, —C(O)CH═CH—,        —CH═CHC(O)—, —(CH₂)_(s1)—, —C(O)—, —C(O)O—, —OC(O)—, or a        valence bond; wherein s1 is 0 or 1.

-   10. The conjugate of any one of embodiments 6-9, wherein W has the    formula

—W₇—Y—,

wherein

-   -   Y is —(CH₂)₁₇—C₃₋₁₀-Cycloalkyl-W₈— or a valence bond,    -   I7 is 0-6,    -   W₇ is selected from —C(O)NH—, —NHC(O)—, —C(O)NHCH₂—,        —CH₂NHC(O)—, —C(O)NHS(O)₂—, —S(O)₂NHC(O)—, —OC(O)NH—, —NHC(O)O—,        —C(O)CH₂—, —CH₂C(O)—, —C(O)CH═CH—, —CH═CHC(O)—, —(CH₂)_(s3)—,        —C(O)—, —C(O)O—, —OC(O)—, or a valence bond; wherein s3 is 0 or        1,    -   W₈ is selected from —C(O)NH—, —NHC(O)—, —C(O)NHCH₂—,        —CH₂NHC(O)—, —C(O)NHS(O)₂—, —S(O)₂NHC(O)—, —OC(O)NH—, —NHC(O)O—,        —C(O)CH₂—, —CH₂C(O)—, —C(O)CH═CH—, —CH═CHC(O)—, —(CH₂)_(s4)—,        —C(O)—, —C(O)O—, —OC(O)—, or a valence bond; wherein s4 is 0 or        1.

-   11. The conjugate of any one of embodiments 6-10, wherein I1, I2,    I3, I4, I5 and I6 independently are 0-6.

-   12. The conjugate of any one of embodiments 6-11, wherein m1, m3,    m4, m6 and m7 independently are 0-6.

-   13. The conjugate of any one of embodiments 6-12, wherein m2 and m5    independently are 0-10.

-   14. The conjugate of any one of embodiments 6-13, wherein n1, n2, n3    and n4 independently are 0-10, such as 0-6.

-   15. The conjugate of any one of embodiments 6-14, wherein D1 and D2    are independently selected from —O— or —NR⁶— or a valence bond.

-   16. The conjugate of any one of embodiments 6-15, wherein D1 is    —NR⁶—.

-   17. The conjugate of any one of embodiments 6-16, wherein D2 is    —NR⁶—.

-   18. The conjugate of any one of embodiments 6-17, wherein D1 and D2    are both —O—.

-   19. The conjugate of any one of embodiments 6-18, wherein D1 and D2    are both —NR⁶—.

-   20. The conjugate of any one of embodiments 6-19, wherein E1 and E2    are independently selected from —O— or —NR⁶— or a valence bond.

-   21. The conjugate of any one of embodiments 6-20, wherein E1 is    —NR⁶—.

-   22. The conjugate of any one of embodiments 6-21, wherein E2 is    —NR⁶—.

-   23. The conjugate of any one of embodiments 6-22, wherein E1 and E2    are both —O—.

-   24. The conjugate of any one of embodiments 6-23, wherein E1 and E2    are both —NR⁶—.

-   25. The conjugate of any one of embodiments 6-24, wherein W₁ through    W₈ independently are selected from the group consisting of —C(O)NH—,    —NHC(O)—, —C(O)NHCH₂—, —CH₂NHC(O)—, —C(O)NHS(O)₂—, —S(O)₂NHC(O)— or    a valence bond.

-   26. The conjugate of any one of embodiments 6-25, wherein R¹, R²,    R³, R⁴ and R⁵ independently are selected from hydrogen, —C(O)OH,    —C(O)NH₂, —S(O)₂OH or C₁₋₆-alkyl; wherein the alkyl group optionally    is substituted with —C(O)OH, —C(O)NH₂, —S(O)₂OH.

-   27. The conjugate of any one of embodiments 6-26, wherein    —{[(CH₂)_(n1)E1]_(m2)-[(CHR²)_(I2)—W₃]_(m3)}_(n2)— and

-   If {[(CH₂)_(n3)E2]_(m5)-[(CHR⁴)_(I4)—W₅]_(m6)}_(n4)—, wherein E1 and    E2 are 0, are selected from

-   -   wherein * is intended to denote a point of attachment, ie, an        open bond.

-   28. The conjugate of any one of embodiments 6-27, wherein A is    selected from

wherein * denotes the attachment to B through W.

-   29. The conjugate of any one of embodiments 1-28, wherein the    albumin binding residue via a hydrophilic spacer is attached to the    glutamine residue in the position corresponding to postion 40 in SEQ    ID No. 1.-   30. The conjugate of any one of embodiments 1-29, wherein the    albumin binding residue via a hydrophilic spacer is attached to the    glutamine residue in the position corresponding to postion 141 in    SEQ ID No. 1.-   31. The conjugate of any one of embodiments 1-30, wherein the    albumin binding residue via a hydrophilic spacer is attached to the    N-terminal residue of the growth hormone compound, such as hGH (SEQ    ID No. 1).-   32. A growth hormone conjugate of the formula (II):

A-W-B-GH-B′-W′-A′  (II)

wherein

-   GH represents a growth hormone compound,-   W is a chemical group linking A and B,-   W′ is a chemical group linking A′ and B′,-   A and A′ independently represents an albumin binding residue,-   B and B′ independently are hydrophilic spacers, and-   pharmaceutically acceptable salts, solvates and prodrugs thereof.-   33. The conjugate of embodiment 32, wherein GH represents a growth    hormone compound comprising an amino acid sequence having at least    90% identity to the amino acid sequence of human growth hormone    (hGH) (SEQ ID NO:1).-   34. The conjugate of embodiment 33, wherein GH is hGH (SEQ ID NO:1).-   35. The conjugate of any one of embodiments 32-34, wherein W′ is    selected from W, A′ is selected from A and B′ is selected from B.-   36. The conjugate of any one of embodiments 32-35, wherein W and W′,    A and A′, and B and B′ independently are selected from their    respective definitions of any one of embodiments 6-31.-   37. The conjugate of any one of the previous embodiments, wherein    the molar weight of said hydrophilic spacer is in the range from 80D    to 1500D or in the range from 500D to 1100D.-   38. The conjugate of any one of the previous embodiments, wherein    said albumin binding residue is a lipophilic residue.-   39. The conjugate of any one of the previous embodiments, wherein    said albumin binding residue binds non-covalently to albumin.-   40. The conjugate of any one of the previous embodiments, wherein    said albumin binding residue is negatively charged at physiological    pH.-   41. The conjugate of any one of the previous embodiments, wherein    said albumin binding residue has a binding affinity towards human    serum albumin that is below about 10 μM or below about 1 μM.-   42. The conjugate of any one of the previous embodiments, wherein    said albumin binding residue is selected from a straight chain alkyl    group, a branched alkyl group, a group which has an ω-carboxylic    acid group or an ω-carboxylic acid isoster.-   43. The conjugate of any one of embodiments the previous    embodiments, wherein said albumin binding residue has from 6 to 40    carbon atoms, from 8 to 26 carbon atoms or from 8 to 20 carbon    atoms.-   44. The conjugate of any one of the previous embodiments, wherein    said albumin binding residue is a peptide, such as a peptide    comprising less than 40 amino acid residues.-   45. The conjugate of any one of the previous embodiments, wherein    said compound is selected from

-   46. The conjugate of any one of embodiments 1-45 for use in therapy.-   47. The conjugate of any one of embodiments 1-45 for use in a method    of treating Turner Syndrome; Prader-Willi syndrome (PWS); Noonan    syndrome; Down syndrome; chronic renal disease, juvenile rheumatoid    arthritis; cystic fibrosis, HIV-infection in children receiving    HAART treatment (HIV/HALS children); short children born short for    gestational age (SGA); short stature in children born with very low    birth weight (VLBW) but SGA; skeletal dysplasia; hypochondroplasia;    achondroplasia; idiopathic short stature (ISS); GHD in adults;    fractures in or of long bones, such as tibia, fibula, femur,    humerus, radius, ulna, clavicula, matacarpea, matatarsea, and digit;    fractures in or of spongious bones, such as the scull, base of hand,    and base of food; patients after tendon or ligament surgery in e.g.    hand, knee, or shoulder; patients having or going through    distraction oteogenesis; patients after hip or discus replacement,    meniscus repair, spinal fusions or prosthesis fixation, such as in    the knee, hip, shoulder, elbow, wrist or jaw; patients into which    osteosynthesis material, such as nails, screws and plates, have been    fixed; patients with non-union or mal-union of fractures; patients    after osteatomia, e.g. from tibia or 1^(st) toe; patients after    graft implantation; articular cartilage degeneration in knee caused    by trauma or arthritis; osteoporosis in patients with Turner    syndrome; osteoporosis in men; adult patients in chronic dialysis    (APCD); malnutritional associated cardiovascular disease in APCD;    reversal of cachexia in APCD; cancer in APCD; chronic abstractive    pulmonal disease in APCD; HIV in APCD; elderly with APCD; chronic    liver disease in APCD, fatigue syndrome in APCD; Crohn's disease;    impaired liver function; males with HIV infections; short bowel    syndrome; central obesity; HIV-associated lipodystrophy syndrome    (HALS); male infertility; patients after major elective surgery,    alcohol/drug detoxification or neurological trauma; aging; frail    elderly; osteo-arthritis; traumatically damaged cartilage; erectile    dysfunction; fibromyalgia; memory disorders; depression; traumatic    brain injury; subarachnoid haemorrhage; very low birth weight;    metabolic syndrome; glucocorticoid myopathy; short stature due to    glucucorticoid treatment inchildren, the acceleration of the healing    of muscle tissue, nervous tissue or wounds; the acceleration or    improvement of blood flow to damaged tissue; or the decrease of    infection rate in damaged tissue.-   48. A pharmaceutical composition comprising a conjugate of any one    of embodiments 1-45, optionally in combination with a pharmaceutical    acceptable excipient.-   49. A method of preparation of a conjugate of any one of embodiments    1-45, the method comprising introduction of an albumin binder linker    to recombinant produced growth hormone or growth hormone analogue    via the TGase method.-   50. A method of treating growth hormone deficiency (GHD), the method    comprising administrating to a patient in need thereof an effective    amount of a therapeutivcally effective amount of a conjugate of any    one of embodiments 1-45.-   51. A method of treating Turner Syndrome; Prader-Willi syndrome    (PWS); Noonan syndrome; Down syndrome; chronic renal disease,    juvenile rheumatoid arthritis; cystic fibrosis, HIV-infection in    children receiving HAART treatment (HIV/HALS children); short    children born short for gestational age (SGA); short stature in    children born with very low birth weight (VLBW) but SGA; skeletal    dysplasia; hypochondroplasia; achondroplasia; idiopathic short    stature (ISS); GHD in adults; fractures in or of long bones, such as    tibia, fibula, femur, humerus, radius, ulna, clavicula, matacarpea,    matatarsea, and digit; fractures in or of spongious bones, such as    the scull, base of hand, and base of food; patients after tendon or    ligament surgery in e.g. hand, knee, or shoulder; patients having or    going through distraction oteogenesis; patients after hip or discus    replacement, meniscus repair, spinal fusions or prosthesis fixation,    such as in the knee, hip, shoulder, elbow, wrist or jaw; patients    into which osteosynthesis material, such as nails, screws and    plates, have been fixed; patients with non-union or mal-union of    fractures; patients after osteatomia, e.g. from tibia or 1^(st) toe;    patients after graft implantation; articular cartilage degeneration    in knee caused by trauma or arthritis; osteoporosis in patients with    Turner syndrome; osteoporosis in men; adult patients in chronic    dialysis (APCD); malnutritional associated cardiovascular disease in    APCD; reversal of cachexia in APCD; cancer in APCD; chronic    abstractive pulmonal disease in APCD; HIV in APCD; elderly with    APCD; chronic liver disease in APCD, fatigue syndrome in APCD;    Crohn's disease; impaired liver function; males with HIV infections;    short bowel syndrome; central obesity; HIV-associated lipodystrophy    syndrome (HALS); male infertility; patients after major elective    surgery, alcohol/drug detoxification or neurological trauma; aging;    frail elderly; osteo-arthritis; traumatically damaged cartilage;    erectile dysfunction; fibromyalgia; memory disorders; depression;    traumatic brain injury; subarachnoid haemorrhage; very low birth    weight; metabolic syndrome; glucocorticoid myopathy; short stature    due to glucucorticoid treatment inchildren, the acceleration of the    healing of muscle tissue, nervous tissue or wounds; the acceleration    or improvement of blood flow to damaged tissue; or the decrease of    infection rate in damaged tissue, the method comprising    administrating to a patient in need thereof an effective amount of a    therapeutivcally effective amount of a conjugate of any one of    embodiments 1-45.-   52. The use of a conjugate of any one of embodiments 1-45 in the    manufacture of a medicament for the treatment of growth hormone    deficiency (GHD).-   53. The use of a conjugate of any one of embodiments 1-45 in the    manufacture of a medicament for the treatment of Turner Syndrome;    Prader-Willi syndrome (PWS); Noonan syndrome; Down syndrome; chronic    renal disease, juvenile rheumatoid arthritis; cystic fibrosis,    HIV-infection in children receiving HAART treatment (HIV/HALS    children); short children born short for gestational age (SGA);    short stature in children born with very low birth weight (VLBW) but    SGA; skeletal dysplasia; hypochondroplasia; achondroplasia;    idiopathic short stature (ISS); GHD in adults; fractures in or of    long bones, such as tibia, fibula, femur, humerus, radius, ulna,    clavicula, matacarpea, matatarsea, and digit; fractures in or of    spongious bones, such as the scull, base of hand, and base of food;    patients after tendon or ligament surgery in, e.g., hand, knee, or    shoulder; patients having or going through distraction oteogenesis;    patients after hip or discus replacement, meniscus repair, spinal    fusions or prosthesis fixation, such as in the knee, hip, shoulder,    elbow, wrist or jaw; patients into which osteo-synthesis material,    such as nails, screws and plates, have been fixed; patients with    non-union or mal-union of fractures; patients after osteatomia,    e.g., from tibia or 1^(st) toe; patients after graft implantation;    articular cartilage degeneration in knee caused by trauma or    arthritis; osteoporosis in patients with Turner syndrome;    osteoporosis in men; adult patients in chronic dialysis (APCD);    malnutritional associated cardiovascular disease in APCD; reversal    of cachexia in APCD; cancer in APCD; chronic abstractive pulmonal    disease in APCD; HIV in APCD; elderly with APCD; chronic liver    disease in APCD, fatigue syndrome in APCD; Crohn's disease; impaired    liver function; males with HIV infections; short bowel syndrome;    central obesity; HIV-associated lipodystrophy syndrome (HALS); male    infertility; patients after major elective surgery, alcohol/drug    detoxification or neurological trauma; aging; frail elderly;    osteo-arthritis; traumatically damaged cartilage; erectile    dysfunction; fibromyalgia; memory disorders; depression; traumatic    brain injury; subarachnoid haemorrhage; very low birth weight;    metabolic syndrome; glucocorticoid myopathy; short stature due to    glucucorticoid treatment inchildren, the acceleration of the healing    of muscle tissue, nervous tissue or wounds; the acceleration or    improvement of blood flow to damaged tissue; or the decrease of    infection rate in damaged tissue.

The description herein of any aspect or embodiment of the inventionusing terms such as “comprising”, “having”, “including” or “containing”with reference to an element or elements is intended to provide supportfor a similar aspect or embodiment of the invention that “consists of”,“consists essentially of”, or “substantially comprises” that particularelement or elements, unless otherwise stated or clearly contradicted bycontext (e.g., a composition described herein as comprising a particularelement should be understood as also describing a composition consistingof that element, unless otherwise stated or clearly contradicted bycontext).

This invention includes all modifications and equivalents of the subjectmatter recited in the aspects or claims presented herein to the maximumextent permitted by applicable law.

The present invention is further illustrated by the following exampleswhich, however, are not to be construed as limiting the scope ofprotection. The features disclosed in the foregoing description and inthe following examples may, both separately and in any combinationthereof, be material for realising the invention in diverse formsthereof.

EXAMPLES Abbreviations:

-   amu=atomic mass units-   hr(s)=hour(s)-   Hz=hertz-   L=liter(s)-   M=molar-   mbar=millibar-   mg=milligram(s)-   min=minute(s)-   mL=milliliter(s)-   mM=millimolar-   mm=milimeter(s)-   mmol=millimole(s)-   nmol=nanomole(s)-   mol=mole(s)-   N=normal-   nm=nanometer(s)-   sec=second(s)-   ppm=parts per million-   ESI=electrospray ionization-   i.v.=intravenous-   m/z=mass to charge ratio-   MS=mass spectrometry-   HPLC=high pressure liquid chromatography-   RP=reverse phase-   HPLC-MS=high pressure liquid chromatography—mass spectrometry-   NMR=nuclear magnetic resonance spectroscopy-   p.o.=per oral-   rt or RT=room temperature-   s.c.=subcutaneous-   tr=retention time-   Boc=tert butyloxycarbonyl-   OtBu=tert butyl ester-   tBu=tert butyl-   Boc-4-ABZ-OH=4-tert-Butoxycarbonylamino-benzoic acid-   DCM=dichloromethane, CH₂Cl₂, methylenechloride-   DIC=diisopropylcarbdiimide-   DIPEA=N,N-diisopropylethylamine-   DMF=N,N-dimethylformamide-   DMSO=dimethylsulfoxide-   DTT=dithiothreitol-   EDAC=1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride-   Et₂O=diethyl ether-   EtOAc=ethyl acetate-   Fmoc=9H-fluoren-9-ylmethoxycarbonyl-   Fmoc-Glu-O-t-Bu=N-Fmoc-glutamic acid-1-t-butyl ester-   Fmoc-Lys(Mtt)-OH═(S)-6-[(Diphenyl-p-tolyl-methyl)-amino]-2-(9H-fluoren-9-ylmethoxycarbonylamino)-hexanoic    acid-   Fmoc-OEG-OH=(2[2-(Fmoc-amino)ethoxy]ethoxy)acetic acid-   H₂O=water-   HOBt=1-hydroxybenzotriazole-   MeCN=acetonitrile-   MeOH=methanol-   NaCl=sodium chloride-   NaOH=sodium hydroxide-   NMP=N-methylpyrrolidin-2-one-   OEG=(2[2-(amino)ethoxy]ethoxy)acetic acid-   TFA=trifuloroacetic acid-   THF=tetrahydrofuran-   TIS=triisopropylsilane-   CDCl₃=deuterio chloroform-   CD₃OD=tetradeuterio methanol-   DMSO-d₆=hexadeuterio dimethylsulfoxide

The examples also make use of the following general methods:

General Method for Preparing a hGH Compounds.

The gene coding for the growth hormone compound was insertedrecombinantly into a plasmid vector. A suitable E.coli strain wassubsequently transformed using the plasmid vector. hGH or GH variantsmay be expressed with an N-terminal methionine or as a MEAE fusion fromwhich the MEAE sequence is subsequently cleaved off.

Cell stock was prepared in 25% glycerol and stored at −80° C. Glycerolstock strain was inoculated into LB plates and subsequently incubated at37° C. overnight. The content of each plate was washed with LB mediumand diluted into 500 mL LB medium for expression. The cultures wereincubated at 37° C. with shaking at 220 rpm until OD₆₀₀ 0.6 was reached.Succeeding induction was performed using 0.2 mM IPTG at 25° C. for 6hrs. Cells were finally harvested by centrifugation.

Cells were subsequently suspended in 10 mM Tris-HCl, pH=9.0 containing0.05% Tween 20, 2.5 mM EDTA, 10 mM cysteamine and 4M urea, and disruptedusing a cell disrupter at 30 kPSI. The supernatant was collected bycentrifugation and subsequently subjected to chromatographicpurification.

The purification was performed using ion-exchange chromatography andhydrophibic interaction, followed by removal of the peptide tag usinghuman dipeptidyl peptidase I (hDPPI) expressed from CHO cell. Finalpurification was achieved by isoprecipitation and ion-exchangechromatography. The purification could also be achieved by using but notlimited to ion-exchange chromatography, hydrophobic interactionchromatography, affinity chromatography, size exclusion chromatographyand membrane based separation techniques known to a person skilled inthe art.

Protein Chemical Characterization of Purified Growth Hormone Compounds.

The intact purified protein was analysed using MALDI-MS. The observedmass corresponded to the theoretical mass deduced from the amino acidsequence. The expected linkage disulfide bonds may be demonstrated bypeptide mapping using trypsin and AspN digestion followed by MALDI-MSanalysis of the digest before and after reduction of the disulfide bondswith DTT.

Capillary Electrophoresis

Capillary electrophoresis was carried out using an Agilent Technologies3DCE system (Agilent Technologies). Data acquisition and signalprocessing were performed using Agilent Technologies 3DCE ChemStation.The capillary was a 64.5 cm (56.0 cm efficient length) 50 μm i.d.“Extended Light Path Capillary” from Agilent. UV detection was performedat 200 nm (16 nm Bw, Reference 380 nm and 50 nm Bw). The runningelectrolyte was phosphate buffer 50 mM pH 7 (method A). The capillarywas conditioned with 0.1 M NaOH for 3 min, then with Milli-Q water for 2min and with the electrolyte for 3 min. After each run, the capillarywas flushed with milli-Q water for 2 min, then with phosphoric acid for2 min, and with milli-Q water for 2 min. The hydrodynamic injection wasdone at 50 mbar for 4.0 sec. The voltage was +25 kV. The capillarytemperature was 30° C. and the runtime was 10.5 min.

Maldi-Tof Mass Spectrometry

Molecular weights were determined using the Autoflex Maldi-Tofinstrument (Bruker). Samples were prepared usingalfa-cyano-4-hydroxy-cinnamic acid as matrix.

RP-HPLC

RP-HPLC analysis was performed on an Agilent 1100 system using a Vydac218TP54 4.6 mm×250 mm 5 μm C-18 silica column (The Separations Group,Hesperia). Detection was by UV at 214 nm, 254 nm, 280 nm and 301 nm. Thecolumn was equilibrated with 0.1% trifluoracetic acid/H₂O and the samplewas eluted by a suitable gradient of 0 to 90% acetonitrile against 0.1%trifluoracetic acid /H₂O.

LC-MS

LC-MS analysis was performed on a PE-Sciex API 100 or 150 massspectrometer equipped with two Perkin Elmer Series 200 Micropumps, aPerkin Elmer Series 200 auto-sampler, a Applied Biosystems 785A UVdetector and a Sedex 75 Evaporative Light scattering detector. A WatersXterra 3.0 mm×50 mm 5μ C-18 silica column was eluted at 1.5 ml/min atroom temperature. It was equilibrated with 5% MeCN/0.1% TFA/H₂O andeluted for 1.0 min with 5% MeCN/0.1% TFA/H₂O and then with a lineargradient to 90% MeCN/0.1% TFA/H₂O over 7 min. Detection was by UVdetection at 214 nm and Evaporative light Scattering. A fraction of thecolumn eluate was introduced into the ionspray interface of a PE-SciexAPI 100 mass spectrometer. The mass range 300-2000 amu was scanned every2 seconds during the run.

Quantification of Protein

Protein concentrations were estimated by measuring absorbance at 280 nmusing a NanoDrop ND-1000 UV-spectrofotometer.

Enzymatic Peptide Mapping for Determination of Site(s) of Derivatization

Peptide mapping was performed using Asp-N digestion of the reduced andalkylated protein. First the protein was treated with DTT andiodoacetamide according to standard procedures. The alkylated productwas purified using HPLC. Subsequently the alkylated purified product wasdigested overnight with endoprotease Asp-N (Boehringer) at anenzyme:substrate ratio of 1:100. The digest was HPLC separated using aC-18 column and standard TFA/MeCN buffer system. The resulting peptidemap was compared to that of underivatized hGH and fractions withdifferent retention times were collected and further analyzed usingMaldi-tof mass spectrometry.

SDS Page

SDS poly-acrylamide gel electrophoresis was performed using NuPAGE4%-12% Bis-Tris gels (Invitrogen NP0321BOX). The gels were silverstained (Invitrogen LC6100) or Coomassie stained (Invitrogen LC6065) andwhere relevant also stained for PEG with barium iodide as described byM. M. Kurfurst in Anal. Biochem. 200(2), 244-248, (1992).

Protein Chromatography

Protein chromatography was performed on an Akta Explorer chromatographicsystem and columns from GE Health Care. Anion exchange was done using aQ-Sepharose HP 26/10 column. Starting buffer was 20 mM triethanolaminebuffer pH 8.5 and eluting buffer was starting buffer +0.2 M NaCl. Thecompounds were typically eluted with a gradient of 0-75% eluting bufferover 15 column volumes. De-salting and buffer exchange was performedusing a HiPrep 26/10 column.

The TGase used in the examples is microbial transglutaminase fromStreptoverticillium mobaraense according to U.S. Pat. No. 5,156,956.

LogP Calculation

LogP values can be calculated as mLogP and/or cLogP for the albuminbinder part and/or the hydrophilic spacer part using publishedalgorithms (J. Am. Chem. Soc., 86 (1964) 5175-5180 “A New SubstituentConstant, ∉, Derived from Partition Coefficients”, C. A. Lipinski et al.Advanced Drug Delivery Reviews, 23 (1997) 3-25, “Experimental andComputational Approaches to Estimate Solubility and Permeability in DrugDiscovery and Development Settings” and I. Moriguchi, S. Hirono, I.Nakagome, H. Hirano, Chem. and Pharm. Bull., 42 (1994) 976-978“Comparison of Reliability of logP Values for Drugs Calculated bySeveral Methods”. Herein clogP—Pomona College logP (octanol/waterpartition coefficient) is calculated with Sybyl 7.0 from Tripos(http://www.tripos.com) version 4.2 of the clogP algorithm and version22 of its associated fragment database as provided by BioByte Corp(http://www.biobyte.com/).

Assay (I) BAF-3GHR Assay to Determine Growth Hormone Activity

The BAF-3 cells (a murine pro-B lymphoid cell line derived from the bonemarrow) was originally IL-3 dependent for growth and survival. 11-3activates JAK-2 and STAT which are the same mediators GH is activatingupon stimulation. After transfection of the human growth hormonereceptor the cell line was turn into a growth hormone-dependent cellline. This clone can be used to evaluate the effect of different growthhormone samples on the survival of the BAF-3GHR.

The BAF-3GHR cells are grown in starvation medium (culture mediumwithout growth hormoen) for 24 hours at 37° C., 5% CO₂.

The cells are washed and re-suspended in starvation medium and seeded inplates. 10 μl of growth hormone compound or human growth hormone indifferent concentrations or control is added to the cells, and theplates are incubated for 68 hours at 37° C., 5% CO₂.

AlamarBlue® is added to each well and the cells are then incubated foranother 4 hours. The AlamarBlue® is a redox indicator, and is reduced byreactions innate to cellular metabolism and, therefore, provides anindirect measure of viable cell number.

Finally, the metabolic activity of the cells is measure in afluorescence plate reader. The absorbance in the samples is expressed in% of cells not stimulated with growth hormone compound or control andfrom the concentration-response curves the activity (amount of acompound that stimulates the cells with 50%) can be calculated.

Assay for Measuring Rate of Protease Degradation of GH and hGH CompoundConjugates

The compound of interest is digested by a relevant protease (Trypsin,Chymotrypsin, Pepsin, Elastase, Factor VIIa, Factor Xa, Proteinase K,Carboxy peptidase, DPPIV, Neutral Endopeptidase, Granzyme B,Proline-endopeptidase, Staphylococcal peptidase I, Thermolysin,Thrombin, Arg-C proteinase, Asp-N endopeptidase, Caspase 1-10,Clostripain, Enterokinase, Glutamyl endopeptidase, Granzyme B, LysC,LysN, Proline-endopeptidase and Staphylococcal peptidase I or tissueextracts.) in an appropriate buffer (e.g. PBS or ammonium bicarbonate)at 37° C. for up till 24 hrs. Proteolytic degradation is assessed by aHPLC assay.

Proteolytic Digestion:

100 μL of test compound solution at 1 mg/mL in ammonium bicarbonatebuffer is degraded by enzyme for up till 24 hrs at 37° C. Sub-samplesare taken to various time points and the proteolytic reaction is stoppedby acidifying the sample by 10 times dilution into 1% TFA. These dilutedsamples are analysed by reversed phase HPLC to estimate the degree ofproteolytic digestion.

HPLC Method:

10 μL of the above solution is injected on a reversed phase Vydac C42x150 mm column eluted with a linear gradient from 0.1% TFA in water to100% acetonitrile containing 0.1% TFA over a period of 30 min at a flowrate of 0.2 ml/min. Detection of peaks is performed at 214 nm UVabsorption. Percentage (%) intact compound at time point t=T iscalculated from the peak area at time point t=T (A_(T)) and the peakarea at t=0 (A₀) as (A_(T)/A₀)×100%. Percentage (%) intact compound isplotted against time using GraphPad Prims software ver. 5.01. Half life(T%) is calculated as one phase decay also by GraphPad Prism software.Examples of enzymes that may be used are elastase (Sigma from porcinepancrease) and chymotrypsin (Roche sequencing grade). Example of bufferis 50 mM ammonium bicarbonate, pH=8.5.

Pharmacokinetics

The pharmacokinetic of the compounds of the examples is investigated inmale Sprague Dawley rats after intravenous (i.v.) and/or subcutaneous(s.c.) single dose administration.

Test compounds are diluted to a final concentration of 1 mg/mL in adilution buffer consisting of: Glycine 20 mg/mL, mannitol 2 mg/mL,NaHCO₃ 2.5 mg/mL, pH adjusted to 8.2.

The test compounds are studied in male Sprague Dawley rats weighing 250g. The test compounds are administered as a single injection either i.v.in the tail vein or s.c. in the neck with a 25 G needle at a dose of 60nmol/kg body weight.

For each test compound blood sampling is conducted according to thefollowing schedule presented in table 1.

TABLE 1 Blood sampling schedule for each test compound. Sampling time(h) Animal no. RoA Predose 0.08 0.25 0.5 1 2 4 6 8 18 24 48 72 1 s.c. XX X X X X 2 X X X X X X 3 X X X X 4 X X X X 5 X X 6 X X 7 i.v. X X X X XX X 8 X X X X X X X 9 X X X 10 X X X

At each sampling time 0.25 mL blood is drawn from the tail vein using a25 G needle. The blood is sampled into a EDTA coated test tube andstored on ice until centrifugation at 1200×G for 10 min at 4° C. Plasmais transferred to a Micronic tube and stored at -20° C. until analysis.

Test compound concentrations are determined by a sandwich ELISA using aguinea pig anti-hGH polyclonal antibody as catcher, and biotinylated hGHbinding-protein (soluble part of human GH receptor) as detector. Thelimit of detection of the assay was 0.2 nM.

A non-compartmental pharmacokinetic analysis is performed on meanconcentration-time profiles of each test compound using WinNonlinProfessional (Pharsight Inc., Mountain View, Calif., USA). Thepharmacokinetic parameter estimates of terminal half-life (t_(%)) andmean residence time (MRT) are calculated.

In vivo dose-response study in hypophysectomised Sprague Dawley rats Thein vivo dose-response relationship is studied in hypophysectomised maleSprague Dawley rats. The hypophysectomised rat is a well known andrecognised animal model of growth hormone deficiency, where noproduction of growth hormone occurs after the surgical removal of thepituitary gland. This also leads to low circulating levels ofinsulin-like growth factor-1 (IGF-1) another important clinical featureof growth hormone deficiency in humans.

The hypophysectomy is performed on 4 week old male rats weighing 90-100g. The animals entering the study 3-4 weeks after the surgery weighing100-110 g. Animals with a body weight gain of more than 10% during the3-4 weeks after surgery are not allowed to enter the study.

Dose response studies are usually performed using five dose levels oftest compound from 1-150 nmol/rat.

Disapperance

Disapperence rates may be meaussered in pigs, usually at least fivefemale pigs of crossbred LYD. The pigs are weighted, fasted and issued aspecial “pig coat” in order to carry the gamma counter and transmitterand placed in single pens before the start of the study. All pigs arefasted for 18 hours prior the study.

The animals are dosed (60 nmol) subcutaneously on the left and the rightside of the neck respectively with a Novopen3® and a NovoFine® 28Gneedle with fixed black needle stopper. Injection depth is 5 mm. Thetest solutions (including the compounds) are diluted in a bufferconsisting of: Glycine 20 mg/mL, mannitol 2 mg/mL, NaHCO₃ 2.4 mg/mL, pHadjusted to 8.2.

Iodination with ¹²⁵1 is performed by Chemistry & Isotope Lab. NovoNordisk A/S. The final radioactive formulation has a specificradioactive activity of 3 μCi/mL and is provided in 3 mL Penfills. Thesolutions were stored at 2-8° C. until used.

The desapperence of the radioactive depots is measured by portableequipment for about 24-48 hours.

Preparation of Albumin Binders Example 1 Tetrazol OEG Linker (I):

The tetrazole OEG linker (I) was synthesised according to scheme 1.

2 g of Rink-Amide-Resin (2 g, 0.6 mMol/g) was weighed into a flask. Theresin was swelled with NMP (3×30 mL) for 2 hrs.

Removal of the Fmoc-group: The resin was shaken with 25% piperidine inNMP (30 mL) for 10 min. The resin was drained and treated with 25%piperidine in NMP (30 mL) for 1 hour followed by draining and wash withNMP (6×30 mL).

Fmoc-Lys(Mtt)-OH and HOBT were weighed into a flask, dissolved in bromophenol blue in NMP (30 mL, 0.5 mM). This solution was added to thedrained resin above followed by addition of DIC. The reaction was shakenat ambient temperature for 21 hrs. The resin was drained and washed withNMP (6×30 mL) followed by washing with DCM (3×30 mL).

The resin was treated with hexafluorisopropanol (20 mL) for 10 min.Shaken for 10 min. The resin was drained and washed with DCM (3×30 mL).The resin was treated with hexafluorisopropanol (20 mL) for 10 min againand shaked for 10 min. The resin was drained and washed with DCM (3×30mL) followed by drained and washed with NMP (3×30 mL).

Boc-4-ABZ-OH and HOBT were weighed into a flask, dissolved in bromophenol blue in NMP (30 mL, 0.5 mM). This solution was added to thedrained resin above followed by addition of DIC. The reaction was shakenat ambient temperature. The resin was drained and washed with NMP (6×30mL).

Removel of the Fmoc-group: The resin was shaken with 25% piperidine inNMP (10 mL) for 10 min. The resin was drained and treated with 25%piperidine in NMP (10 mL) for 1 hour followed by draining and wash withNMP (6×15 mL).

Fmoc-OEG-OH and HOBT were weighed into a flask, dissolved in brom phenolblue in NMP (15 mL, 0.5 mM). This solution was added to the drainedresin followed by addition of DIC. The reaction was shaken at ambienttemperature for 23 hrs. The resin was drained and washed with NMP (6×15mL).

Removal of the Fmoc-group: The resin was shaken with 25% piperidine inNMP (10 mL) for 10 min. The resin was drained and treated with 25%piperidine in NMP (10 mL) for 1 hour followed by draining and wash withNMP (6×15 mL).

Fmoc-Glu-O-t-Bu and HOBT were weighed into a flask, dissolved in bromophenol blue in NMP (15 mL, 0.5 mM). This solution was added to thedrained resin followed by addition of DIC. The reaction was shaken atambient temperature for 18 hrs. The resin was drained and washed withNMP (6×15 mL).

Removal of the Fmoc-group: The resin was shaken with 25% piperidine inNMP (10 mL) for 10 min. The resin was drained and treated with 25%piperidine in NMP (10 mL) for 1 hour followed by draining and wash withNMP (6×15 mL).

Fmoc-Glu-O-t-Bu and HOBT were weighed into a flask, dissolved in 15 ml0.5 mM bromo phenol blue in NMP. This solution was added to the drainedresin followed by addition of DIC. The reaction was shaken at ambienttemperature for 18 hrs. The resin was drained and washed with NMP (6×15mL).

Removal of the Fmoc-group: The resin was shaken with 25% piperidine inNMP (10 mL) for 10 min. The resin was drained and treated with 25%piperidine in NMP (10 mL) for 1 hour followed by draining and washingwith NMP (6×15 mL).

Fmoc-OEG-OH and HOBT were weighed into a flask, dissolved in bromophenol blue in NMP (15 mL, 0.5 mM). This solution was added to thedrained resin followed by addition of DIC. The reaction was shaken atambient temperature. The resin was drained and washed with NMP (6×15mL).

Removal of the Fmoc-group: The resin was shaken with 25% piperidine inNMP (10 mL) for 10 min. The resin was drained and treated with 25%piperidine in NMP (10 mL) for 1 hour followed by draining and washingwith NMP (6×15 mL).

4-(16-1H-Tetrazol-5-yl-hexadecanoylsulfamoyl)-butyric acid and HOBT wereweighed into a flask, dissolved in bromo phenol blue in NMP (15 mL, 0.5mM). This solution was added to the drained resin followed by theaddition of DIC. The reaction was shaken at ambient temperature for 21hrs. The resin was drained and washed with NMP (6×15 mL NMP) followed bydraining and wash with DCM (6×15 mL).

The resin was cleaved with a mixture of 95% TFA in water (10 mL)+DCM(0,25 mL) and TIPS (0.25 mL). The resin was shaken for 2 hrs at ambienttemperature and filtered into cold diethyl ether (75 mL). The resultingprecipitate was isolated by centrifugation followed by washing withdiethyl ether (3×) and dried in vacuum for 48 hrs affording crude 300 mgof the compound. TOF-MS: Rt=4,7 min, mass 1268.71

The crude compound was purified on prep-HPLC (GILSON). T2145-10; 30->80%MeCN. Pooled fractions were evaporated to dryness on rotavap and theresidue dissolved in H₂O/MeCN 1:1 and freezedried over night affording170 mg of the compound.

Example 2

In a similar way as described in Example 1 above the following compoundmay be prepared using FMOC-Lys(Mtt)-OH and Wang Resin.

Example 3

In a similar way as described in Example 1 above and depicted below thefollowing compound was prepared using Boc-Gly-PAM resin as startingmaterial.

Example 4

In a similar way as described in Example 1 above the following compoundwas prepared using FMOC-Lys(Mtt)-OH and Rink amid resin.

Example 5

In a similar way as described in Example 1 above the following compoundwas prepared using FMOC-Lys(Mtt)-OH and Rink amid resin.

Example 6

In a similar way as described in Example 1 above the following compoundwas prepared using FMOC-Lys(Mtt)-OH and Rink amid resin.

Example 7

In a similar way as described in Example 1 above the following compoundwas prepared using FMOC-Lys(Mtt)-OH and Rink amid resin.

Example 8

In a similar way as described in Example 1 above the following compoundwas prepared using FMOC-Lys(Mtt)-OH and Rink amid resin.

Example 9

In a similar way as described in Example 1 above the following compoundwas prepared using FMOC-Lys(Mtt)-OH and Wang Resin.

Example 10

In a similar way as described in Example 1 above the following compoundwas prepared using FMOC-Lys(Mtt)-OH and Rink amid resin.

Example 11

In a similar way as described in Example 1 above the following compoundwas prepared using FMOC-Lys(Mtt)-OH and Rink amid resin.

Example 12

In a similar way as described in Example 1 above the following compoundwas prepared using FMOC-Lys(Mtt)-OH and Wang Resin.

Example 13

In a similar way as described in Example 1 above the following compoundwas prepared using FMOC-Lys(Mtt)-OH and Rink amid resin.

Example 14

In a similar way as described in Example 1 above the following compoundmay be prepared using FMOC-Lys(Mtt)-OH and Rink amid resin.

Example 15

In a similar way as described in Example 1 above the following compoundmay be prepared using FMOC-Lys(Mtt)-OH and Rink amid resin.

Example 16

In a similar way as described in Example 1 above the following compoundmay be prepared using FMOC-Lys(Mtt)-OH and Rink amid resin.

Example 17

In a similar way as described in Example 1 above the following compoundwas prepared using FMOC-Lys(Mtt)-OH and Rink amid resin.

Example 18

In a similar way as described in Example 1 above the following compoundwas prepared using FMOC-Lys(Mtt)-OH and Rink amid resin.

Example 19

In a similar way as described in Example 1 above the following compoundwas prepared using FMOC-Lys(Mtt)-OH and Rink amid resin.

Example 20

In a similar way as described in Example 1 above the following compoundmay be prepared using FMOC-Lys(Mtt)-OH and Rink amid resin.

Example 21

In a similar way as described in Example 1 above the following compoundmay be prepared using FMOC-Glu(ODmab)-OH and 2-chlorotrityl chlorideresine.

Example 22

In a similar way as described in Example 1 above the following compoundmay be prepared using FMOC-Glu(ODmab)-OH and 2-chlorotrityl chlorideresine.

Example 23

In a similar way as described in Example 1 above the following compoundwas prepared using FMOC-Lys(Mtt)-OH and Rink amid resin.

Example 24

In a similar way as described in Example 1 above the following compoundmay be prepared using FMOC-Lys(Mtt)-OH and Rink amid resin.

Example 25

In a similar way as described in Example 1 above the following compoundmay be prepared using FMOC-Glu(ODmab)-OH and 2-chlorotrityl chlorideresine.

Preparation of GH Albumin Binder Compounds Example 26

1. Coupling of Albumin Binder of General formular A-W-B1-NH2 toTransaminated and Oxidised GH compound (I)

The use of a transglutaminase to attach an aldehyde handle to GH onglutamine residues has previously been described in WO2005/070468. Themethod may be used in accordance with the present invention forattachment of a albumin binder based linker of formula A-W-B1-NH2. TheTGase used is microbial transglutaminase from Streptoverticilliummobaraense according to U.S. Pat. No. 5,156,956.

The reaction may be performed as follows: GH (I) is initiallytransaminated with 1,3-diamino-2-propanol (II) as described inWO2005/070468:

In the next step, transaminated GH (III) is added periodate. Theoxidation is typically done at low temperature, such as 4-10° C. over 30min. optionally in the dark. Periodate may oxidize metheonine residuesin GH to their corrosponding metheonine sulfoxide residues. To minimizethis oxidation risk, small molecule organic thioethers may be addedduring periodate oxidation. A suitable organic thioether is3-methylthiopropan-1-ol but the skilled person will be able to suggestothers.

Oxidation of Transaminated GH Compound (Ill):

Buffer change may be performed in order to obtain an acid solutionrequired for efficient sodium cyano borohydride reduction. Typically, anexcess of A-W-B1-NH2 amine is used, and sodium cyanoborohydride may beadded in smaller portions over time.Reductive amination of (IV) with albumin binder linker (V):

The later reaction may be performed as follows:

A solution of oxidized transaminated GH (IV) is added a solution ofalbumin binder linker (V) in a mixture of AcOH (1,5 mL) and 50 mM MES(0.5 mL) at pH 6.00. The resulting reaction mixture is gently shaken atRT for 30 min. at which time a NaCNBH₃ solution (15 μL, (22 mg NaCNBH₃dissolved in 500 μL Milli-Q water+AcOH (15 μL))) is added. The sample iscovered with tin foil and stirrer over night at RT.

The conjugate can be isolated by anion exchange chromatography asfollows: Acetic acid is removed by buffer changed with pure water (3×)using Amicon Ultra15 devices (Ultracel 10K tubes) by centrifugation at4000 rpm/min. for 3×8 min. The mixture is then buffer changed to 20 mMTEA, pH: 8.50 using Amicon Filter devises and diluted to a final volumeof 50 mL with 20 mM TEA, before loading it on a HiLoad Q Sepharose,26/10 column. The column is initially washed with 20 mM TEA, pH 8.50(buffer A) and then eluted with 20 mM TEA, 500 mM NaCl, pH 8.50 (bufferB) using a 0-100%(B) gradient over 20 CV, with a flow rate of 2 ml/min.The pooled fractions were buffer changed 5 times to 10 mMammoniumbicarbonate buffer in pure water using Amicon Ultra15 devices(Ultracel 10K tubes) by centrifugation at 4000 rpm/min. for 3×8 min

Example 27

In a similar way as described in Example 26 above the following compoundmay be prepared using albumin binder from Example 2.

Example 28

In a similar way as described in Example 26 above the following compoundwas prepared using albumin binder from Example 4.

Example 29

In a similar way as described in Example 26 above the following compoundwas prepared using albumin binder from Example 5.

Example 30

In a similar way as described in Example 26 above the following compoundwas prepared using albumin binder from Example 6.

Example 31

In a similar way as described in Example 26 above the following compoundwas prepared using albumin binder from Example 7.

Example 32

In a similar way as described in Example 26 above the following compoundwas prepared using albumin binder from Example 8.

Example 33

In a similar way as described in Example 26 above the following compoundwas prepared using albumin binder from Example 9.

Example 34

In a similar way as described in Example 26 above the following compoundwas prepared using albumin binder from Example 10.

Example 35

In a similar way as described in Example 26 above the following compoundwas prepared using albumin binder from Example 11.

Example 36

In a similar way as described in Example 26 above the following compoundwas prepared using albumin binder from Example 12.

Example 37

In a similar way as described in Example 26 above the following compoundwas prepared using albumin binder from Example 13.

Example 38

In a similar way as described in Example 26 above the following compoundmay be prepared using albumin binder from Example 14.

Example 39

In a similar way as described in Example 26 above the following compoundmay be prepared using albumin binder from Example 15.

Example 40

In a similar way as described in Example 26 above the following compoundmay be prepared using albumin binder from Example 16.

Example 41

In a similar way as described in Example 26 above the following compoundwas prepared using albumin binder from Example 17.

Example 42

In a similar way as described in Example 26 above the following compoundwas prepared using albumin binder from Example 18.

Example 43

In a similar way as described in Example 26 above the following compoundmay be prepared using albumin binder from Example 19.

Example 44

In a similar way as described in Example 26 above the following compoundmay be prepared using albumin binder from Example 20.

Example 45

In a similar way as described in Example 26 above the following compoundmay be prepared using albumin binder from Example 21.

Example 46

In a similar way as described in Example 26 above the following compoundmay be prepared using albumin binder from Example 22.

Example 47

In a similar way as described in Example 26 above the following compoundwas prepared using albumin binder from Example 23.

Example 48

In a similar way as described in Example 26 above the following compoundmay be prepared using albumin binder from Example 24.

Example 49

In a similar way as described in Example 26 above the following compoundmay be prepared using albumin binder from Example 25.

Example 50

Coupling of albumin binder linker of general formular A-W-B1-NH₂ totransaminated and oxidised GH compound (I)

The use of a transglutaminase to attach an aldehyde handle to GH onglutamine residues has previously been described in WO2005/070468. Themethod may be used in accordance with the present invention forattachment of a albumin binder based linker of formula A-W-B1-NH2. TheTGase used is microbial transglutaminase from Streptoverticilliummobaraense according to U.S. Pat. No. 5,156,956.

The reaction may be performed as follows: GH (I) is initiallytransaminated with 1,3-diamino-2-propanol (II) as described inWO2005/070468:

Transaminated hGH Gln⁴⁰ (Ill) was obtained from Example 26 as abiproduct from the CIE chromatography purification.

In the next step, transaminated GH (III) is added periodate. Theoxidation is typically done at low temperature, such as 4-10° C. over 30min. optionally in the dark. Periodate may oxidize metheonine residuesin GH to their corrosponding metheonine sulfoxide residues. To minimizethis oxidation risk, small molecule organic thioethers may be addedduring periodate oxidation. A suitable organic thioether is3-methylthiopropan-1-ol but the skilled person will be able to suggestothers.

Oxidation of transaminated GH compound (III):

Buffer change may be performed in order to obtain an acid solutionrequired for efficient sodium cyano borohydride reduction. Typically, anexcess of A-W-B1-NH2 amine is used, and sodium cyanoborohydride may beadded in smaller portions over time.

Reductive amination of (IV) with albumin binder linker (V):

The later reaction may be performed as follows:

A solution of oxidized transaminated GH (IV) is added a solution of bileacid linker (V) in a mixture of AcOH (1.5 mL) and 50 mM MES (0.5 mL) atpH 6.00. The resulting reaction mixture is gently shaken at RT for 30min. at which time a NaCNBH₃ solution (15 μL, (22 mg NaCNBH₃ dissolvedin 500 μL Milli-Q water+AcOH (15 μL))) is added. The sample is coveredwith tin foil and stirrer over night at RT.

The conjugate can be isolated by anion exchange chromatography asfollows: Acetic acid is removed by buffer changed with pure water (3X)using Amicon Ultra15 devices (Ultracel 10K tubes) by centrifugation at4000 rpm/min. for 3×8 min. The mixture is then buffer changed to 20 mMTEA, pH: 8.50 using Amicon Filter devises and diluted to a final volumeof 50 mL with 20 mM TEA, before loading it on a HiLoad Q Sepharose,26/10 column. The column is initially washed with 20 mM TEA, pH 8.50(buffer A) and then eluted with 20 mM TEA, 500 mM NaCl, pH 8.50 (bufferB) using a 0-100%(B) gradient over 20 CV, with a flow rate of 2 mL/min.The pooled fractions were buffer changed 5 times to 10 mMammoniumbicarbonate buffer in pure water using Amicon Ultra15 devices(Ultracel 10K tubes) by centrifugation at 4000 rpm/min. for 3×8 min

In a similar way as described in Example 50 above the following compoundwas prepared using albumin binder from Example 6.

Example 51

1. Coupling of a GH Compound (I) N-Terminaly with an Albumine Binder(II)

(A) Reductive Alkylation of (I) with an Albumin Binder Aldehyde (II)

Albumin binder (II) was obtained as described in Example 3.

2-C₂₀diacid-Trx-γGlu-Glu-OEG-OEG-Gly-Glycin amid)-ethyl-N^(α1)-hGH

hGH (23 mg) was dissolved in Hepes buffer (2.3 mL 0.25 mM pH 7,0).C₂₀diacid-Trx-yGlu-Glu-OEG-OEG-Gly-Gly-dimethylacetal (2 mg, see example3 above) was treated with TFA (50 μL) for 6 min. and evaporated todryness in vacuum. The residue was stripped with EtOH (200 μL) andevaporated to dryness in vacuum. The residue was dissolved in DMF (100μL) and added to the hGH solution. A precipitate was formed andredissolved by addition of DMF (1 mL). After 1 hr a solution of NaCNBH₃(20 mg, in 0.5 mL MeCN (230 μL)) was added portionwise and left for 20hrs. The reaction was quenched by addition of AcOH (2 mL) and dilutedwith water to a total volume of 20 ml and purified on prep. HPLC on aC18 column with a gradient of MeCN/0.1% TFA from 40-80% against 0.1% TFAin water. The latest eluting peak was collected, diluted from 70% MeCNto 10% with water and lyophilized affording 4,51 mg of the compound.TOF-MS: mass 23.237,6

Example 52

GH compounds as described herein were tested in one or more of theassays described herein above.

EC50 values based on BAF assay as well as half life (T%) and meanresidence time (MRT) were determined and included in table 2 below.

Disapperence rate for selected compounds were meaussered in five femalepigs of crossbred LYD as described above. The result are presented asAUC (0-45 hrs) in table 2. The solubility enhancing propertiy expressedas cLogP of the space (B) as determined by the method described above isalso included in table 2.

Albumin binding affinity as determined by Biacor and or HPLC is alsoincluded in table 2.

Data relating to a pegylated hGH compound is included as well. PEG-hGHbeing identical to the compound disclosed in Example 1 ofWO2006024953A2.

TABLE 2 Solublity Albumin of T_(1/2) Mean Albumin binding Albumin spacerEC50/ (i.v. residence Disapperance binder (HPLC) binder and (B)¹ potencyRat) time AUC affinity (Rt, Compound attachment cLogP (BAF) (hours)(MRT) (0-45 hour) (Biacor) min) hGH — — 1 0.15 —  519 — 0.36 hGH-PEG — —26 5.8 — 2426 — — Ex. 28 Gln141 −5.32 1.4 1.6 8.3 ND 0.58 7.16 Ex. 4 Ex.29 Gln141 0.25 1.3 1.5 12.3  1659 0.50 6.76 Ex. 5 Ex. 30 Gln141 −6.791.5 2 3.6 2283 0.76 7.00 Ex. 6 Ex. 31 Gln141 −7.05 1.7 ND ND 1977 0.657.46 Ex. 7 Ex. 32 Gln141 −0.44 0.3 ND ND 1500 ND 4.25 Ex. 8 Ex. 33Gln141 −2.17 0.63 ND ND 1789 0.02 6.57 Ex. 9 Ex. 34 Gln141 −0.44 0.620.84 ND  903 ND 6.01 Ex. 10 Ex. 35 Gln141 −3.43 0.78 1.6 7.0 ND 0.236.75 Ex. 11 Ex. 36 Gln141 −2.43 0.84 1.8 7.4 1606 0.41 6.88 Ex. 12 Ex.37 Gln141 −5.07 0.64 0.86 1.5 1260 ND 6.45 Ex. 13 Ex. 41 Gln141 −2.742.52 2.3 6.2 1570 0.29 7.06 Ex. 17 Ex. 42 Gln141 −4.76 2.10 ND ND ND ND5.69 Ex. 18 Ex. 47 Gln141 −0.67 ND ND ND ND ND 7.00 Ex. 23 Ex. 50 Gln40−6.79 1.60 2.8 4.3 ND 0.55 ND Ex. 6 Ex. 51 N-term −5.87 2.80 2.5 11.6 1935 0.74 7.07 Ex. 3 ¹For calculation purpose * has been set to carbonin the individual B-spacer elements.

1. A growth hormone conjugate which comprises a growth hormone compound(GH) linked to an albumin binding residue via a hydrophilic spacer, or apharmaceutically acceptable salt, solvate or prodrug thereof.
 2. Theconjugate of claim 1 wherein the growth hormone conjugate has theformula (I):A-W-B-GH   (I) wherein: GH is a growth hormone compound, B is ahydrophilic spacer, W is a chemical group linking A and B, and A is analbumin binding residue;
 3. The conjugate of claim 1, wherein GH is hGH(SEQ ID NO:1).
 4. The conjugate of claim 2 wherein B has the formula—X₁—X₂—X₃—X₄— wherein X₁ is—W₁-[(CHR¹)_(I1)—W₂]_(m1)—{[CH₂)_(n1)E1]_(m2)—[(CHR²)₁₂—W₃]_(m3)}_(n2)—;X₂ is—[(CHR³)₁₃—W₄]_(m4)—{[(CH₂)_(n3)E2]_(m5)—[(CHR⁴)₁₄—W₅]_(m6)}_(n4)—; X₃is —[(CHR⁵)₁₅—W₆]_(m7)—; X₄ is F-D1-(CH₂)_(I6)-D2-; I1, I2, I3, I4, I5and I6 independently are selected from 0-16; m1, m3, m4, m6 and m7independently are selected from 0-10; m2 and m5 independently areselected from 0-25; n1, n2, n3 and n4 independently are selected from0-16; F is aryl, hetaryl, pyrrolidine-2,5-dione or a valence bond,wherein the aryl and hetaryl groups are optionally substituted withhalogen, —CN, —OH, —C(O)OH, —C(O)NH₂, —S(O)₂OH or C₁₋₆-alkyl; R¹, R²,R³, R⁴ and R⁵ independently are selected from hydrogen, —C(O)OH,—C(O)NH₂, —S(O)OH, —S(O)₂OH, —NH—C(═NH)—NH₂, C₁₋₆-alkyl, aryl orhetaryl; wherein the alkyl, aryl and hetaryl groups optionally aresubstituted with halogen, —C(O)OH, —C(O)NH₂, —S(O)OH, —S(O)₂OH, —CN or—OH; D1, D2, E1 and E2 independently are selected from —O—, —NR⁶—,—N(COR⁷)— or a valence bond, wherein R⁶ and R⁷ independently representhydrogen or C₁₋₆-alkyl; W₁ to W₆ independently are selected from—C(O)NH—, —NHC(O)—, —C(O)NHCH₂—, —CH₂NHC(O)—, —C(O)NHS(O)₂—,—S(O)₂NHC(O)—, —OC(O)NH—, —NHC(O)O—, —C(O)CH₂—, —CH₂C(O)—, —C(O)CH═CH—,—CH═CHC(O)—, —(CH₂)_(s2)—, —C(O)—, —C(O)O—, —OC(O)—, or a valence bond,wherein s2 is 0 or
 1. 5. The conjugate of claim 2, wherein W has theformula—W₇—Y—, wherein Y is —(CH₂)₁₇-C₃₋₁₀-cycloalkyl-W₈— or a valence bond, 17is 0-6, W₇ is selected from the group consisting of —C(O)NH—, —NHC(O)—,—C(O)NHCH₂—, —CH₂NHC(O)—, —C(O)NHS(O)₂—, —S(O)₂NHC(O)—, —OC(O)NH—,—NHC(O)O—, —C(O)CH₂—, —CH₂C(O)—, —C(O)CH═CH—, —CH═CHC(O)—, —(CH₂)_(s3)—,—C(O)—, —C(O)O—, —OC(O)—, or a valence bond, wherein s3 is 0 or 1; W₈ isselected from the group consisting of —C(O)NH—, —NHC(O)—, —C(O)NHCH₂—,—CH₂NHC(O)—, —C(O)NHS(O)₂—, —S(O)₂NHC(O)—, —OC(O)NH—, —NHC(O)O—,—C(O)CH₂—, —CH₂C(O)—, —C(O)CH═CH—, —CH═CHC(O)—, —(CH₂)—, —C(O)—,—C(O)O—, —OC(O)—, or a valence bond, wherein s4 is 0 or
 1. 6. Theconjugate of claim 4, wherein I1, I2, I3, I4, I5 and I6 independentlyare 0-6, m1, m3, m4, m6 and m7 independently are 0-6, m2 and m5independently are 0-10, and n1, n2, n3 and n4 independently are 0-10. 7.The conjugate of claim 4, wherein D1 and D2 are independently selectedfrom —O— or —NR⁶— or a valence bond.
 8. The conjugate of claim 4,wherein E1 and E2 are independently selected from —O— or —NR⁶— or avalence bond.
 9. The conjugate of claim 4, wherein W₁ through W₈independently are selected from the group consisting of —C(O)NH—,—NHC(O)—, —C(O)NHCH₂—, —CH₂NHC(O)—, —C(O)NHS(O)₂—, —S(O)₂NHC(O)— and avalence bond.
 10. The conjugate of claim 4, wherein R¹, R², R³, R⁴ andR⁵ independently are selected from the group consisting of hydrogen,—C(O)OH, —C(O)NH₂, —S(O)₂OH and C₁₋₆-alkyl, wherein the alkyl groupoptionally is substituted with —C(O)OH, —C(O)NH₂, or —S(O)₂OH.
 11. Theconjugate of claim 2, wherein A is selected from

wherein * denotes the attachment to B through W.
 12. The conjugate ofclaim 1, wherein the albumin binding residue via a hydrophilic spacer isattached to the glutamine residue in the position corresponding toposition 40 in SEQ ID No. 1, or the glutamine residue in the positioncorresponding to position 141 in SEQ ID No. 1, or the N-terminal residueof the growth hormone compound.
 13. The conjugate of claim 1, whereinsaid compound is selected from the group consisting of


14. A pharmaceutical composition comprising a conjugate of claim1,optionally in combination with a pharmaceutically acceptableexcipient.
 15. A method of treating one a disease or condition treatablewith an increased amount of circulating growth hormone comprisingadministering an effective amount of the growth hormone conjugate ofclaim
 1. 16. The method of claim 15 wherein the disease or condition isselected from the group consisting of growth hormone deficiency (GHD),Turner Syndrome, Prader-Willi syndrome (PWS), Noonan syndrome andidiopathic short stature (ISS).